Maf 55013 1 ap

Cells were washed twice in PBS and protein extracts prepared in radioimmunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF, 1% protease inhibitor mixture P8340, and 1% phosphate inhibitor mixtures 2 and 3; all Sigma-Aldrich). Equal total amounts of protein were resolved on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (MilliporeSigma) using a Bio-Rad SD Semi-Dry Transfer Cell, blocked for 2 h at room temperature in 2% BSA (Thermo Fisher Scientific) or 5% milk powder (Sigma-Aldrich) in TBST (150 mM NaCl, 7.7 mM Tris HCl [pH 8], and 0.1% Tween 20; all Sigma-Aldrich) before overnight probing with primary Abs at 4°C. Abs were as follows: Maf (55013-1-AP) and Stat4 (51070-2-AP) from Proteintech, Histone 3 from Cell Signaling Technology. Following extensive washing in TBST, blots were incubated with secondary Abs (goat anti-rabbit or mouse HRP; DAKO) for 1 h at room temperature, washed as before, and developed with ECL Western Blotting Detection Reagent and Hyperfilm ECL (both GE Healthcare).