2.4. Cell lysis, immunoprecipitation, and Western blot analyses

For whole‐cell lysate collection, cells were washed with phosphate‐buffered saline and lysed in lysis buffer (50 mM HEPES (pH 7.5), 150 mM KCl, 5 mM EDTA, 0.05% IGEPAL CA‐630 (v/v), 1× protease inhibitor cocktail (Roche) and 1× phosphatase inhibitor cocktail (Cell Signaling Technology). Following sonication and centrifugation, total protein yield was determined by Bicinchoninic Acid (BCA) Protein assay (Sigma‐Aldrich). Total protein (20 µg) samples were denatured in 1× Laemmli Buffer supplemented with 8% β‐mercaptoethanol for 5 min at 95°C.

For immunoprecipitation, protein samples were prepared at 1 µg/mL protein in lysis buffer. Lysates were incubated overnight with 3 µg of FLAG antibody (Cat#F1804, Sigma‐Aldrich), or INTS3 antibody (Cat# A302‐050 Bethyl) at 4°C. Following incubation, lysates were incubated for 1 h with protein A or G Dynabeads (Thermo Fisher Scientific) pre‐equilibrated with lysis buffer. The Dynabeads were denatured using 2× Laemmli sample buffer supplemented with 8% β‐mercaptoethanol for 5 min at 95°C.

Samples were separated on Bolt 4%–12% Bis‐Tris Plus pre‐cast gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membrane (GE Healthcare Life Sciences) using the semi‐dry transfer Novex system (Thermo Fisher Scientific). Membranes were first blocked using Odyssey blocking buffer (Li‐Cor) and then incubated with primary antibody overnight at 4°C in a 1:1 solution of Odyssey blocking buffer and PBS‐T. RNA Pol II CTD (clone 4H8, cat# 2629) and Androgen Receptor (D6F11, cat# 5153) antibodies were from Cell Signaling Technology and hSSB1 antibody was raised in‐house as described previously. ³⁴ All primary antibodies were used at a dilution of 1:1000. Following incubation, membranes were washed with PBS‐T and incubated with appropriate secondary antibodies and imaged using the Li‐Cor Odyssey system (Li‐Cor).