Abstract
INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation.
Introduction
T
Recently it has been reported that INI1/hSNF5/BAF47 plays a dual role by antagonizing the expression of the genes that are inhibited or activated by OCT4 during differentiation in human embryonic NCCIT cells.(9) To further elucidate the role of INI1/hSNF5/BAF47 during cell differentiation, we generated a specific monoclonal antibody against INI1/hSNF5/BAF47.
Materials and Methods
Cell culture
Human cervical carcinoma HeLa cells were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified atmosphere of 5% CO2 at 37°C.
Production and purification of recombinant proteins
A full-length INI1/hSNF5/BAF47-glutathione S-transferase (GST) fusion protein expression vector and GST protein expression vector were transformed into Escherichia coli BL21(DE3) (Novagen, Madison, WI). The E. coli cells were grown in LB medium containing 50 μg/mL carbenicillin (Nacalai Tesque, Kyoto, Japan) at 37°C. Expression, bacteria lysis and purification of the fusion protein were performed as previously described.(7)
Mouse immunization and production of monoclonal antibody
The anti-INI1/hSNF5/BAF47 mouse monoclonal antibody was generated based on the rat lymph node method established by Sado and colleagues.(11,12) A 6-week-old female ICR mouse (SLC, Shizuoka, Japan) was injected in the hind footpads with 150 μL of an emulsion containing 125 μg of INI1/hSNF5/BAF47 protein fused to GST and Freund's complete adjuvant. After 2 weeks, the cells from the medial iliac lymph nodes of this mouse were fused with mouse myeloma SP2 cells at a ratio of 5:1 in a 50% polyethylene glycol (PEG 1500, Roche, Mannheim, Germany) solution. The resulting hybridoma cells were plated in 96-well plates and cultured in HAT selection medium (Hybridoma-SFM [Invitrogen, Carlsbad, CA], 10% FBS, 10% BM condimed H1 [Roche, Mannheim, Germany], 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine). At 7 days post-fusion, the hybridoma supernatants were screened by an enzyme-linked immunosorbent assay (ELISA) against the INI1/hSNF5/BAF47-GST protein. Positive clones were subcloned and rescreened by ELISA and immunoblotting. The specific 2C2 MAb immunoglobulin class was determined using a mouse isotyping kit. The analysis indicated that the MAb 2C2 is a mouse IgG2a (kappa).
Immunoblotting/CBB stain
Whole cell extracts (WCE) of HeLa cells and recombinant protein GST-Ini1/GST were separated by 10% or 15% SDS-PAGE and electrophoretically transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA). The membranes were blocked for 1 h at room temperature (RT) with a blocking solution containing 3% skim milk for WCE and 5% skim milk for recombinant proteins in TBS-T (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% Tween-20), and then incubated for 1 h at RT with anti-INI1/hSNF5/BAF47 mouse MAb 2C2 (2C2) diluted in the blocking solution for detecting from WCE, and 2C2 and anti GST antibodies diluted in the Signal Enhancer Hikari (Nacalai Tesque) for detecting from recombinant protein and GST protein. After washing with TBS-T, the membranes were incubated for 1 h at RT with alkaline phosphatase-conjugated anti-mouse IgG antibody (Sigma, St Louis, MO) for WCE and with horseradish peroxidase-conjugated anti-mouse IgG antibody (GE Healthcare, Little Chalfont, UK). Following washes with TBS-T, the signals were detected by treatment with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for WCE and treatment with 5-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione (luminol) for recombinant proteins. Recombinant proteins were electrophoresed by another 15% SDS-PAGE gel; then that gel was fixed for 10 min in CBB destaining solution (5% ethanol, 7.5% acetate). Following this step, gel was stained for 1 h in CBB staining solution (50% methanol, 10% acetate, 0.2% CBB) and destained by CBB destaining solution for 30 min.
Immunocytochemistry
HeLa cells grown on coverslips were washed twice with PBS and fixed with 3.7% formaldehyde for 15 min at RT. After rapid washing with PBS, cells were permeabilized for 5 min in 0.5% Triton X-100 in PBS and then incubated for 30 min in blocking buffer. INI1/hSNF5/BAF47 was detected with MAb 2C2, followed by an Alexa 488-conjugated goat anti-mouse IgG (Invitrogen) for 1 h at RT. Coverslips were mounted with Prolong (Molecular Probes, Carlsbad, CA). INI1/hSNF5/BAF47-expressing cells were visualized by fluorescence microscopy (Olympus, Tokyo, Japan).
Results and Discussion
Two isoforms of INI1/hSNF5/BAF47 have been identified: long and short. The long one has 385 amino acids, and the short one has 376 amino acids. We attempted to produce a specific monoclonal antibody against INI1/hSNF5/BAF47 using a full-length GST fusion-protein of the long isoform as the antigen and the mouse lymph node method. To this end, 2C2, a hybridoma that produces a specific monoclonal antibody against INI1/hSNF5/BAF47, was established. The heavy chain isotype of this antibody is IgG2a and the light chain is kappa. The ability to recognize INI1/hSNF5/BAF47 of the established 2C2 clone was tested by immunoblotting. The antibody produced by 2C2 strongly reacted as a double-band at ∼45 kDa with a total cell extract of HeLa cells (Fig. 1A). The expected molecular weight of INI1/hSNF5/BAF47 is 43 kDa in the short isoform and 44 kDa in the long isoform. Therefore, the monoclonal antibody against INI1/hSNF5/BAF47 recognized two isoforms. For evaluation of specificity of this antibody, immunoblotting and CBB staining were performed using purified recombinant INI1/hSNF5/BAF47-GST fusion protein and GST (Fig 1B). The result showed that 2C2 antibody recognized INI1 specifically. In addition, it is known that INI1/hSNF5/BAF47 is a nuclear protein. Immunostaining to assess the nuclear localization of INI1/hSNF5/BAF47 protein confirmed its nuclear localization (Fig. 2). These results demonstrate that the monoclonal antibody against INI1/hSNF5/BAF47 produced by 2C2 can recognize INI1/hSNF5/BAF47 in immunoblotting and immunostaining. Moreover, the immunostaining capability of this antibody suggests that it may be used for ChIP analysis as well.
The monoclonal antibody produced by 2C2 can detect INI1/hSNF5/BAF47 specifically. (
INI1/hSNF5/BAF47 is localized in the nucleus in HeLa cells. The 2C2 MAb confirmed that INI1/hSNF5/BAF47 is localized in the nucleus. HeLa cells were immunostained with the 2C2 MAb. The MAb was detected with Alexa 488-conjugated anti-mouse IgG. The image was counter-stained with Hoechst.
In summary, the usage of this anti-INI1/hSNF5/BAF47 monoclonal antibody may promote the analysis of complexes that contain INI1/hSNF5/BAF47 and their distribution pattern in the genome. Thus, we expect that this antibody will help elucidate the function of INI1/hSNF5/BAF47.
Footnotes
Acknowledgments
We thank Dr. Yoshikazu Sado of Shigei Medical Institute for his advice. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Author Disclosure Statement
Taro Tachibana is a founder of Cell Engineering Corporation.