Monoclonal Antibody That Recognizes a Domain on Heterogeneous Nuclear Ribonucleoprotein K and PTB-Associated Splicing Factor

Abstract

hnRNP K protein is a member of the heterogeneous nuclear protein (hnRNP) complex that, besides its function as a translational regulator of human mRNA, is also considered to be a transcription factor involved in tumorigenesis. PSF is a protein part of the human spliceosome and essential in RNA splicing. Here we report the generation of one monoclonal antibody GG6H9.1C3 that recognized both hnRNP K and PSF proteins using Western blot analysis, flow cytometry, and immunocytochemistry.

Introduction

The nucleus is a complex organelle in which a wide range of processes occur. Specific nuclear factors are assigned to these activities. For example, the splicing factor usually means binding to RNA and participating in RNA splicing activities, while the transcription factor insinuates interactions with DNA and regulation of transcription.

In eukaryotic cells, heterogeneous nuclear RNAs (hnRNAs), from which mRNAs are generated by RNA processing, associate with specific nuclear proteins to form large heterogeneous nuclear protein (hnRNP) complexes. hnRNPs, considered the most important group of splicing factors, repress splicing by directly antagonizing the recognition of splice sites, or can interfere with the binding of proteins bound to enhancers. To date, about 20 hnRNPs (A–U) have been identified. Lately, hnRNPs have attracted considerable interest due to accumulating evidence pointing to their multiplicity of function in mRNA biogenesis, cytoplasmic mRNA trafficking, and participation in almost every step in the life cycle of mRNA, from transcription and processing to transport and translation, including activities such as transcription factors.^(1,2)

Analysis of the hnRNPs has provided strong evidence that most (if not all) of them bind directly to RNA through different structural motifs such as RBD, RNA binding domain, and RGG, arginine/glycine-rich box.^(3,4)

hnRNP K is the major poly(iC)-binding protein; the binding of hnRNP K to nucleic acid is mediated by three repeat motifs called the KH (K homology) domains rather than by the RBD or RGG domains found in all hnRNPs. hnRNP K presents five RGG domains. hnRNP K is abundant in the nucleus but also in the cytoplasm and mitochondria and is implicated in many different chromatin remodelling, transcription, splicing, and translation processes.^(5,6)

hnRNP K not only functions as a translational regulator of human mRNA but also represses expression of regulators of cell cycle control, proliferation, and differentiation—gene regulation, as it is considered a transcription factor.⁽⁷⁾ Its differential localization has also been found to be relevant in tumorigenesis.^(8–10)

PSF, polypyrimidine tract-binding protein (PTB)-associated splicing factor, is another essential pre-mRNA splicing factor. The polypyrimidine tract is a region found in most introns of higher eukaryotes, to which several factors can bind, and is important for the definition of the 3P-splice site. PSF is part of human spliceosome and it was identified and characterized in a complex with PTB. PTB's (also named hnRNP I) role reserves binding sites for essential splicing factors such as PSF. PSF was characterized as a 100 kDa with RNA and DNA-binding properties. PSF has been identified in the nucleolus and in association with the nuclear membrane. Most nuclear proteins interacting with nucleic acids contain either a DNA-binding domain or RNA-binding domain; one exception is PSF and p54^nrb/NonO that possess both domains.⁽¹¹⁾ PSF has an RNA-binding RGG element instead of five like the hnRNP K, and it is probably methylated and therefore cannot bind RNA.⁽¹²⁾

Antibodies that recognize hnRNP K protein or PSF are available from different laboratories and companies. To our knowledge, there is no monoclonal antibody (MAb) available that recognizes an epitope on hnRNP K and PSF protein.

A group of MAbs that recognized proteins from the hnRNP complex were developed using purified poly(A)⁺ material from UV-irradiated HeLa cell nuclei and cytoplasm.⁽¹³⁾ The first specific monoclonal antibodies for the K protein, clone 3C2, were prepared by immunizing BALB/c mice with hnRNPs purified by oligo(dC) affinity chromatography.⁽¹⁴⁾ More recently, other specific MAbs to K protein were produced using a C-terminal peptide or an internal sequence of non-methylated arginine residues conjugated to ovalbumin as immunogens, respectively.^(9,10) Many different monoclonal antibodies against the hnRNP complex, including K protein, are available from Dreyfuss Laboratory (www.med.upenn.edu/dreyfusslab/reagents.html).

Antibodies that recognized PSF are also available, some prepared against PSF peptides 1T25 and 1T13 after coupling to BSA.⁽¹¹⁾ A monoclonal antibody (clone B92) was obtained after immunization of BALB/c mice with cell lysates of an endothelial adipose stromal cell line that, besides a 49 kDa protein, reacts with PSF protein from various organs.⁽¹⁵⁾ Monoclonal antibodies that, as well as the PTB protein, recognized a 100 kDa protein that is probably PSF were obtained using purified recombinant human PTB as an antigen.⁽¹⁶⁾

In our attempt to generate monoclonal antibodies against cell markers of human molecules of HS181, we obtained a group of monoclonal antibodies in which some of them are directed against different nuclear protein. We report here the generation and characterization of MAb GG6H9.1C3, which recognizes an epitope on hnRNP K and PSF proteins, and its capacity to recognize these proteins by various techniques.

Materials and Methods

Cell culture

Human embryonic stem cell line HS181 was cultured as previously described⁽¹⁷⁾ on mitomycin C-inactivated human foreskin fibroblast feeder cells (CRL-2429, ATCC) at 37°C, 5% CO₂ in DMEN/KO, supplemented with 20% knockout serum replacement (Gibco, Carlsbad, CA), 2 mM L-glutamine (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 1% non-essential amino acids (Gibco), and 50 U/mL and 50 mg/mL penicillin and streptomycin, respectively (Gibco). Cells were subcultured every 10 days by mechanical splitting. For the experimental conditions described in this study, the hECS were cultured on Matrigel-coated tissue culture plates (BD Biosciences, San Jose, CA) in the medium described above conditioned with human foreskin fibroblast, in presence of 8 ng/mL bFGF.

Generation of MAbs

Female BALB/c mice (4–6 weeks of age) were immunized by intraperitoneal injection of approximately 15 colonies of HS181 cells. Before the immunization, each colony was torn into a smaller size and suspended in phosphate buffered saline (PBS, pH 7.2). The mice were bled 3 days after the third injection, and the sera were tested for the reactivity to HS181 cells by indirect ELISA. Three days after the last immunization, the splenic lymphocytes were fused with the (SP2/0-AG14) mouse myeloma cell line and hybridomas were selected in RPMI 1640 medium (Lonza, Basel, Switzerland) containing 10% fetal bovine serum, 2 mM L-glutamine (Gibco), 50 U/mL and 50 mg/mL penicillin and streptomycin, respectively (Gibco), and HAT component (Sigma, St. Louis, MO), as described previously.^(18,19) The culture supernatants of hybridomas were tested for reactivity for HS181 by indirect ELISA, and each positive clone was isolated after two steps of subcloning by limiting dilution.

Screening of hybridomas

The resulting hybridomas were screened by an enzyme-linked immunosorbent assay (ELISA). To set up the ELISA, 96-well plates were pre-coated with 5mg/mL of poly-Lysine (Sigma, St. Louis, MO) for 30 min at room temperature (RT). Thereafter, 5.10⁵ of HS181 cell line in 50 μL of PBS were added to the wells and centrifuged at 1500 g for 10 min. After centrifugation, the cells in each well were fixed in 50 μL of 0.5% glutaraldehyde in PBS (Sigma) for 15 min at RT. The plates were then washed twice with PBS and the wells blocked with 200 μL of PBS containing 2% bovine serum albumin (BSA) for 1 h at RT. The plates were washed twice with PBS and then incubated for 1 h at 37°C or overnight at 4°C with 200 μL in each well of supernatant of each antibody or culture medium only. The latter was used as negative reference. After washing three times with PBST (PBS containing 0.05% Tween-20), the well was then incubated with 50 μL of 1:250 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse polyvalent immunoglobulins (Sigma) for 1 h at 37°C. After incubation, the well was washed three times with PBST and then reacted for 1–3 min at RT with 100 μL peroxidase chromogenic substrate (0.5 M citric acid pH4 + 1/100 (v/v) 2% ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [Boehringer Ingelheim, Ingelheim, Germany]) + 0.03% H₂O₂). The reaction was stopped by adding 50 μL of NaF (0.5 M) into each well, and the absorbence of each well at 405 nm was recorded.

Determination of immunoglobulin subtype

Isotype analysis of each antibody was carried out according to the manufacturer's protocol (mouse immunoglobulin isotyping ELISA kit, BD Biosciences, San Jose, CA).

Immunocytochemistry

For immunocytochemistry, the hESC HS181 and Shef-6 on a 12-well plate were washed with Ca2+- and Mg2+-PBS, fixed in 4% paraformaldehyde for 15 min at RT, and permeabilized in 0.3% Triton-X100 in PBS for 15 min at RT. After washing three times in PBS, the plate was blocked with 5% fetal bovine serum in PBS for 1 h at RT. The hESCs were incubated overnight with culture supernatant of each MAb and were detected with Fluorescein (FITC)–conjugated AffiniPure Goat Anti-mouse IgM (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA). SSEA-4 and SSEA-3 were used as positive control for HS181 and Shef-6 cell line, respectively. Next, the cells were stained with 300 nM of 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were visualized through Olympus IX71 microscope (Olympus, Hamburg, Germany).

Flow cytometry

Single cell suspensions for flow cytometry were made from the undifferentiated cells, HS181, Shef-6, Shef-4, H7(S6), H7(S14), and other cell lines, NTERA-2/D1, n2102Ep Cl2A/6, 14H11, P-19. Cells were suspended in FACS buffer (5% fetal bovine serum in PBS) following trypsinization, and 10⁷ cells taken for staining. The cells were fixed in 4% paraformaldehyde for 15 min at RT and permeabilized in 0.3% Triton-X100 in PBS for 15 min at RT. After washing once in FACS buffer, the dissociated cells were incubated with culture supernatant of each monoclonal antibody for 30 min at 4°C with shaking. They were washed three times with FACS buffer and, following the final wash, the cells were resuspended in FACS buffer and incubated with a FITC-conjugated goat anti-mouse secondary antibody (1:100; Caltag, Buckingham, United Kingdom) for 30 min at 4°C with shaking. Cells were washed three times with FACS buffer again, and analyzed on the CyAnADP O2 (Dako, Glostrup, Denmark) flow cytometer. For a negative control, hESCs were stained with secondary antibody only, as described earlier. For a positive control we used anti-SSEA-3,⁽²⁰⁾ anti-SSEA-1,⁽²¹⁾ and TRA1-60.⁽²²⁾

Immunoblot analysis

Cells were lysed in different lysis buffers, RIPA (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), Tris-Triton for cytoskeletal proteins (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1% Triton-X100, 10% glicerol, 0.1% SDS, 0.5% sodium deoxicolate, 1 mM EDTA, 1 mM EGTA), containing 1% protease inhibitor cocktail (Sigma) at 4°C for 1 h. For nucleolar proteins we used the Lamond protocol (www.lamondlab.com). Their protein concentrations were determined using Bio-Rad Protein Assay, based on the method of Bradford. The cell lysates were stored at −70°C before use. For SDS-polyacrylamide gel electrophoresis (PAGE), 20 μg of cell lysate was mixed with sample treatment buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 0.1% bromophenol blue, and 20% β-mercaptoethanol) and boiled before loading in each well. Native PAGE was similar to SDS-PAGE, except that β-mercaptoethanol was omitted in the sample buffer and the mixture did not undergo boiling. Cell lysates were separated on a gradient 5–15% polyacrilamide gel and then transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) using transfer buffer containing 48 mM Tris, 39 mM glycine, 0.038% SDS, and 20% methanol (pH 9.2). The membranes were blocked with 5% dry milk-TBST (20 mM Tris-HCl [pH 7.4], 0.15 M NaCl, 0.05% Tween-20) for 1 h and incubated overnight with each culture supernatant and anti-hnRNP K (1:150) (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. Membranes were washed three times (10 min for each wash) with TBST between labeling steps. For detection of bound murine MAb, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse polyvalent immunoglobulin (Sigma), diluted 1:10.000 with 5% milk-TBST. Finally, the immunoblots were visualized using ECL detection reagent (GE Healthcare, Little Chalfont, United Kingdom).

Protein identification by mass spectrometry (MALDI-TOF/TOF MS)

Bands were isolated manually from SDS-PAGE and digested with modified porcine trypsin (sequencing grade; Promega Biotech Ibérica, Alcobendas, Spain) using a ProGest digestion station (Genomic Solutions, Huntingdon, United Kingdom). Gel pieces were destained twice over 30 min at 37°C with 200 mM ammonium bicarbonate/40% ACN, and subsequently subjected to three consecutive dehydratation/rehydratation cycles with pure ACN and 25 mM ammonium bicarbonate in 50% ACN, respectively. Finally, gel pieces were dehydrated for 5 min with pure ACN and dried over 4 h at room temperature. Then, 20 μL trypsin (12.5 ng/μL in 25 mM ammonium bicarbonate) was added to dried gel pieces and digestion proceeded at 37°C for 12 h. Peptides were extracted from gel plugs by adding 1 μL of 10% (v/v) TFA and incubating for 15 min. Then they were desalted and concentrated by using μC-18 ZipTip columns (Millipore) in a ProMS station (Genomic Solutions) and directly loaded onto the MALDI plate using α-cyano hydroxycinnamic acid as the matrix. Mass analysis of peptides of each sample was performed with a MALDI-TOF/TOF (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA) in automatic mode with the following setting: for the MS data, m/z range 800–4000 with an accelerating voltage of 20 kV and delayed extraction, peak density of maximum 50 peaks per 200 Da, minimal S/N ratio of 10 and maximum peak at 65. Spectra were internally calibrated with peptides from trypsin autolysis (M+H+ = 842.509, M+H+ = 2211.104).

For the MS/MS data, mass range was set between 60 Da and 10 Da below each precursor mass, with a minimum S/N ratio of 5, a maximum number of peaks set at 65 and peak density of maximum 50 peaks per 200 Da. Proteins were assigned identification by peptide mass fingerprinting and confirmed by MS/MS analysis of at least three peptides in each sample. Mascot 1.9 search engine (Matrix Science, London, United Kingdom) was used for protein identification running on GPS software (Applied Biosystems) against the NCBI mammalian and bacteria protein database (updated monthly).

Search setting allowed one missed cleavage with the selected trypsin enzyme, a MS/MS fragment tolerance of 0.2 Da and a precursor mass tolerance of 100 ppm.

Results and Discussion

Of a total of 1632 wells, 1097 (67%) supernatant contained hybridomas and out of a total of 631 hybridomas selected, 62 were shown to produce MAbs binding specifically to HS181 cell line in ELISA analysis. GG6H9.1C3, a monoclonal antibody cloned twice by limiting dilution and with the isotype IgM and kappa light chain, was selected among the group of antibodies that recognized intracellular antigen in human embryonic stem cell lines by immunocytochemistry. Figure 1 shows the antigen recognized by GG6H9.1C3 is localized in cytoplasm and nucleus. We do not have a convincing explanation of the fact that only a percentage of the HS181 cell line was stained by GG6H9.1C3; this was probably due to an inadequate membrane permeabilization or to a culture process since another repeated experiment with HS181 cells offered similar results (Fig. 1A, inset). These results were not confirmed with colonies of the hESC line Shef-6 since all the cells were stained (Fig. 1B).

FIG. 1.

Expression of GG6H9.1C3 in human embryonic stem cell line HS181 (A) and Shef-6 (B) by immunocytochemistry. Colonies were subsequently stained with anti-mouse detection conjugated with FITC and nuclear counterstained with DAPI. Cell colonies from HS181 were stained positive for SSEA-4 and Shef-6 for SSEA-3 (data not shown). Inset, higher magnification of negative, marked with an arrow and positive cells from HS181 stained with GG6H9.1C3. Dapi, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

The cell distribution of the antigen recognized by GG6H9.1C3 was performed by flow cytometry on a different permeabilized human embryonic stem cell line such as HS181, Shef-6, and H7 (S6), and embryonal carcinoma cells such as NTERA-2/D1, n2102Ep, 1411H, and P-19. Figure 2 shows the results obtained by flow cytometry, demonstrating that the antigen was expressed 100% in all cell lines.

FIG. 2.

Analysis of GG6H9.1C3 MAb binding to cell lines by flow cytometry. Representative FACS histograms of GG6H9.1C3 expressing on embryonic stem cell line (HS181, Shef-6, H7) and embryonal carcinoma (NTERA-2/D1, n2102Ep, 1411H, P-19). The specific staining is indicated by black histograms; corresponding isotype negative control is indicated by gray line.

Analysis of GG6H9.1C3 by Western blot analysis on different cell lines showed the recognition of a double band of 65 and 100 kDa on total lysate extracts of human cell lines NTERA-2/D1 and n2102Ep or the mouse embryonic cell line D3 (Fig. 3).

FIG. 3.

Western blot of different cell lines probed with the GG6H9.1C3 monoclonal antibody. Total protein (20 μg) was loaded and separated on a gradient 5–15% reducing SDS-PAGE. Two immunoreactive bands of apparent M_r 100 and 65 kDa were observed. T, total protein extract; NT2, NTERA-2 clone D1.

The GG6H9.1C3 antibody immunoreactive bands with 100-65 kDa were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis to generate peptide mass fingerprints. Mass spectra obtained from MALDI-TOF mass spectrometer were searched against NCBI redundant protein sequence database with the MASCOT search engine. One of the mass spectra is shown in Figure 4 as an example. After database search, the two immunoreactive proteins bands with GG6H9.1C3 antibody of 100 kDa and 65 kDa were successfully identified as the PSF protein (PTB-associated splicing factor) and the hnRNP K protein, respectively. MASCOT score obtained was of 490 and 121 and sequence coverage of 29 and 25%, respectively.

FIG. 4.

Analysis of the GG6H9.1C3 immunoreactive protein with MALDI-TOF/TOF MS. Mass spectra of the immunoreactive PSF protein (PTB-associated splicing factor) digested with trypsin. Total sequence coverage of 29% was achieved. The accession number of the matched protein is gi/119627830.

To confirm the identity of the GG6H9.1C3 antibody immunoreactive protein as hnRNP K, the PVDF membrane was stripped and restained with the commercially available MAb, clone 3C2, specific for hnRNP K protein⁽¹⁴⁾ (Santa Cruz Biotechnology, Santa Cruz, CA), following the protocol of immunoblot analysis described in the Material and Methods section. A coincident protein band was detected by 3C2 and GG6H9.1C3 (Fig. 5).

FIG. 5.

Western blot analysis of total protein extracts of NTERA-2/D1, n2102Ep, and D3 cell lines. Comparison with commercial MAb for hnRNP K and GG6H9.1C3 expression. T, total protein extract; NT2, NTERA-2 clone D1; 1, supernatant MAb GG6H9.1C3; 2, hnRNP K (commercial MAb clone 3C2); PSF, PTB-associated splicing factor.

Since nhRNP K is the only hnRNP protein present in nucleus and cytoplasm, we fractionated different embryonic and carcinoma cell line proteins into nuclear and cytoskeletal fractions to further confirm that the target antigen of GG6H9.1C3 was present in nucleus and cytoplasm. If GG6H9.1C3 in fact recognizes hnRNP K, the immunoreactive signals should be localized in the nuclear and cytoskeletal fraction. Indeed, as shown in Figure 6A, immunoreactive signals corroborate the location of the GG6H9.1C3 antigen in the nuclear and cytoskeletal fraction connected with the cell distribution of hnRNP K.

FIG. 6.

Western blot analysis of different cell lines of nuclear and cytoskeletal proteins by GG6H9.1C3 and hnRNP K clone 3C2 antibodies. 20 μg nuclear and cytoskeletal proteins (20 μg each) were separated in a gradient 5–15% reducing SDS-PAGE and probed with GG6H9.1C3 (A) or hnRNP K (B) antibodies as described in Materials and Methods. Cell line lysates: nuclear and cytoskeletal NTERA-2/D1 lysates, nuclear n2102Ep lysate, and nuclear Hela lysate. NT2, NTERA-2 clone D1; PSF, PTB-associated splicing factor.

The same immunoblot was stripped and stained with the commercial antibody specific to hnRNP K, 3C2 clone. The same band was detected (Fig. 6B). This result further confirmed that GG6H9.1C3 recognized an epitope on the hnRNP K protein (65 kDa), an epitope that is also present on the PSF protein (100 kDa). PSF protein localization differed markedly from the hnRNP K distributions; PSF was present in subnuclear fractions or isolated nuclear matrices and not present in cytoplasm. The weak 100 kDa band on cytoskeletal lysates from NTERA-2/D1 cells is probably due to cytoplasmic contamination.

This monoclonal antibody was obtained as part of an effort to characterize human embryonic stem cells and was obtained using complete human HS181 cell line as immunogen. Unexpectedly, the antibody detects nuclear proteins from humans and mice highly conserved during evolution.

We have produced and characterized a new MAb GG6H9.1C3 that could be very useful to detect hnRNP K and PSF proteins in the nucleus or cytoplasm of different cells and tissues. This antibody can be employed using different techniques such as ELISA, cytometry, immunocytochemistry, or Western blot. With the data offered here, we cannot determine what epitope is recognized by GG6H9.1C3. Our conjecture is that GG6H9.1C3 recognizes a methylated epitope on the RGG domain. RGG domains are present on both proteins; five on hnRNP K and one in PSF, the RGG domain in PSF being methylated.⁽¹²⁾ Comparison with other MAbs against non-methylated epitopes of hnRNP K⁽¹⁰⁾ could provide some light on the characteristics of the epitope recognized by GG6H9.1C3 on hnRNP K and PSF.

Footnotes

Acknowledgments

This work was funded by Fundación Progreso y Salud (FPS), Junta de Andalucía, Spain (D.L.); Instituto de Salud Carlos III (TERCEL RD06/0010/0025) (B.S.); and Fundación Progreso y Salud–Junta de Andalucía (PI-0095/2007) and Instituto de Salud Carlos III (CIBERDEM) (J.R.T.). Also appreciated is funding from Consejería de Salud, Junta de Andalucía (EF-0005/2008) (G.G.). We are especially indebted to Peter Andrews (Sheffield University, United Kingdom) for providing embryonic carcinoma cell lines. We are grateful to the laboratory personnel of the Department of Biomedical Science, Sheffield University, for technical assistance in cytometry analysis. We also thank Nuria Mellado Damas and Yolanda Aguilera García (CABIMER) for technical assistance with human embryonic stem cell line HS181 and Unidad de Proteómica, SCAI, University of Córdoba for the proteomics analysis.

Author Disclosure Statement

The authors have no financial conflicts to declare.

References

Moore

. From birth to death: the complex lives of eukaryotic mRNAs. Science, 2005; 309:1514–1518.

Martinez-Contreras

, Cloutier

, Shkreta

, Fisette

, Revil

, Chabot

. hnRNP proteins and splicing control. Adv Exp Med Biol, 2007; 623:123–147.

Gorlach

, Burd

, Portman

, Dreyfuss

. The hnRNP proteins. Mol Biol Rep, 1993; 18:73–78.

Krecic

, Swanson

. hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol, 1999; 11:363–371.

Mandal

, Vadlamudi

, Nguyen

, Wang

, Costa

, Bagheri-Yarmand

, Mendelsohn

, Kumar

. Growth factors regulate heterogeneous nuclear ribonucleoprotein K expression and function. J Biol Chem, 2001; 276:9699–9704.

Bomsztyk

, Denisenko

, Ostrowski

. hnRNP K: one protein multiple processes. Bioessays, 2004; 26:629–638.

Michelotti

, Michelotti

, Aronsohn

, Levens

. Heterogeneous nuclear ribonucleoprotein K is a transcription factor. Mol Cell Biol, 1996; 16:2350–2360.

Pino

, Pio

, Toledo

, Zabalegui

, Vicent

, Rey

, Lozano

, Torre

, Garcia-Foncillas

, Montuenga

. Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer. Lung Cancer, 2003; 41:131–143.

Carpenter

, McKay

, Dundas

, Lawrie

, Telfer

, Murray

. Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer. Br J Cancer, 2006; 95:921–927.

10.

Naarmann

, Harnisch

, Flach

, Kremmer

, Kuhn

, Ostareck

, Ostareck-Lederer

. mRNA silencing in human erythroid cell maturation: heterogeneous nuclear ribonucleoprotein K controls the expression of its regulator c-Src. J Biol Chem, 2008; 283:18461–18472.

11.

Patton

, Porro

, Galceran

, Tempst

, Nadal-Ginard

. Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev, 1993; 7:393–406.

12.

Shav-Tal

, Zipori

. PSF and p54(nrb)/NonO—multi-functional nuclear proteins. FEBS Lett, 2002; 531:109–114.

13.

Dreyfuss

, Choi

, Adam

. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol, 1984; 4:1104–1114.

14.

Matunis

, Michael

, Dreyfuss

. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Mol Cell Biol, 1992; 12:164–171.

15.

Lee

, Shav-Tal

, Peled

, Gothelf

, Jiang

, Toledo

, Ploemacher

, Haran-Ghera

, Zipori

. A hematopoietic organ-specific 49-kD nuclear antigen: predominance in immature normal and tumor granulocytes and detection in hematopoietic precursor cells. Blood, 1996; 87:2283–2291.

16.

Grossman

, Meyer

, Wang

, Mulligan

, Kobayashi

, Helfman

. The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points. RN, 1998; 4:613–625.

17.

Hovatta

, Mikkola

, Gertow

, Stromberg

, Inzunza

, Hreinsson

, Rozell

, Blennow

, Andang

, Ahrlund-Richter

. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod, 2003; 18:1404–1409.

18.

Llanes

, Nogal

, Prados

, del Val

, Vinuela

. An erythroid species-specific antigen of swine detected by a monoclonal antibody. Hybridoma, 1992; 11:757–764.

19.

Arce

, Moreno

, Millan

, Martin de las Mulas

, Llanes

. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes. Vet Immunol Immunopathol, 2002; 88:31–41.

20.

Shevinsky

, Knowles

, Damjanov

, Solter

. Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell, 1982; 30:697–705.

21.

Solter

, Knowles

. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1) Proc Natl Acad Sci USA, 1978; 75:5565–5569.

22.

Andrews

, Banting

, Damjanov

, Arnaud

, Avner

. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma, 1984; 3:347–361.