Co-immunoprecipitation Assay with Modified Protocol

Co-immunoprecipitation (Co-IP) assays were performed as described previously^{44 (link),45 (link)} with a modified protocol using NETN buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40) containing Protease Inhibitor cocktail (Roche #11836170001) and phosphatase inhibitor cocktail (Roche #4906845001) to extract whole-cell protein, or a cell fractionation protocol for cytoplasmic and nuclear fractionation using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific #78833). Cell lysates were incubated with the indicated antibodies at 4 °C overnight. The protein complex was captured using protein A-agarose or protein G-agarose beads (Roche, Indianapolis, IN, USA) at 4 °C for 4 h, and agarose beads were collected by centrifuge and washed three times with washing buffer. The precipitated proteins were mixed with 1× Blue Loading Buffer along with 3 mM dithiothreitol (Cell Signaling Technology #7722) and subjected to western blot analysis. The following primary antibodies were used for Co-IP: BRD4 (Cell Signaling, Cat#13440, RRID: AB_2687578), HA-tag (Cell Signaling, Cat# 5017, RRID: AB_10693385), Flag-tag (Sigma-Aldrich, Cat#F1804, RRID:AB_262044), and STAT3 (Cell Signaling, Cat#9139, RRID:AB_331757).