2.8. Western Blotting and Immunoprecipitation

Cells were treated with CA, ruxolitinib, or DMSO for 2 h. Pellets were collected and washed with PBS, then lysed with RIPA buffer (Sigma R0278) supplemented with protease inhibitors (Sigma P8340) and phosphatase inhibitors (Sigma P0044 and P5726). Proteins were resolved on NuPAGE 4–12% polyacrylamide gels in LDS buffer (Thermo Fisher), transferred to PVDF membranes with Tris-Glycine transfer buffer, blocked with 5% BSA or 5% milk in 0.1% TBST, then probed with antibodies. Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724). For secondary antibodies, we used either anti-rabbit IgG HRP conjugate (Promega #401B) or anti-mouse IgG HRP conjugate (Promega #402B).