Protein a sepharose 4 fast flow

After washing with PBS, cells (1 × 10⁷) were fixed with 1% formamide in PBS for 10 minutes and incubated with 0.125 mol/L glycine in PBS for 5 minutes. The cells were lysed by RIPA buffer (25 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, and 1% sodium deoxycholate) containing protein inhibitor cocktail (Calbiochem). The lysates were sonicated to fragment genomic DNA into approximately 600‐1000 bp, and centrifuged at 20 630 g for 10 minutes to remove insoluble materials. After transferring the lysates to new tubes, immunoprecipitation was carried out with an anti‐Ap2α Ab (sc‐184; Santa Cruz Biotechnology) and Protein A Sepharose 4 Fast Flow (GE Healthcare). After washing three times with washing buffer (50 mmol/L Tris‐HCl, pH 7.4, 1 mmol/L Na₃VO₄, 150 mmol/L NaCl, and 0.5% Triton X‐100), the immunoprecipitates were heated in 500 µL Tris‐EDTA at 95°C for 15 minutes. Then RNaseA (50 µg/mL; Sigma) treatment at 37°C for 30 minutes and Proteinase K (200 µg/mL; Sigma) treatment at 55°C for 1 hour were carried out. DNA purification was undertaken using phenol and chloroform by the standard method. Finally, ethanol precipitation was carried out to pellet DNAs, and the pellets were dissolved in 50 µL Tris‐EDTA. Using these DNA solutions, PCR and quantitative PCR analyses were carried out. Primers used in this study are listed in Table S2.

SABP1-specifc polyclonal antibodies were obtained by immunization of rabbits and rats with His-SABP1 fusions. The rabbits and rats were subcutaneously immunized 4 times in Freund’s adjuvants. Rabbit–anti-SABP1 IgG was purified from rabbit sera using protein A Sepharose 4 Fast Flow (GE Healthcare).