Ye361

Whole-cell lysates were obtained from mouse mφs at the specified time points by lysing the cells in complete radioimmunoprecipitation lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 2 mM sodium orthovanadate, and 1× protease inhibitor mixture [Fisher, BPE9706-1] in PBS). Phosphatase inhibitor was added to the lysis buffer when harvesting cell lysates for pStat6 detection. Cell lysate (10 μg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blot was incubated with either goat polyclonal anti-Pu.1 (1:200, D-19, sc-5949; Santa Cruz Biotechnology), rabbit monoclonal anti-Stat6 (1:2000, YE361, ab32520; Abcam), or rabbit polyclonal anti-pStat6 (1:1000, Tyr⁶⁴¹, ab195647; Abcam). Protein was detected using ECL (Thermo Fisher Scientific).

STAT6 dimerization experiment was performed with freshly made solution of disuccinimidyl suberate (DSS, Thermo Scientific). At 36hr-posttransfection, cells were harvested and washed three times with ice-cold PBS (pH 8.0) to remove amine-containing culture media and proteins from the cells. Cells pellet was re-suspended in PBS to reach a concentration of 2.5x10⁷ cells per ml, and cross-linked with 1mM DSS for 30 minutes at room temperature. The cross-link reaction was stopped by addition of 20mM Tris-HCl (pH 7.5) and incubated for another 15 min at room temperature, followed by washing with PBS for once and lysis with RIPA for 30min at ice. The cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with STAT6 antibody (YE361, Abcam) for endogenous dimer of STAT6.