Androgen Receptor Interaction with TRPM8 in PC3 Cells

PC3-TRPM8 cells were transfected with peGFP-AR (wild-type, 23 or Q640X). After 48 h of transfection, the cells were incubated with different concentration of testosterone (1, 10 or 100 nM) during 20 min at 37 °C and washed twice with PBS. Cells were incubated on ice in lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM NaKPO₄, pH 7.2, supplemented with anti-protease cocktail; Sigma-Aldrich). After lysate centrifugation (12,000 × g for 10 min at 4 °C), the protein concentration was determined using BCA assay (Thermo Fischer Scientific). An equal amount of supernatants was incubated overnight at 4 °C with 40 µl of His-tag beads (dynadeads® His-tag, Thermo Fischer Scientific) in IP buffer (20 mM NaH₂PO₄, 150 mM NaCl, pH 8). The pellet was washed three times in IP buffer, resuspended in SDS sample buffer, heated at 95 °C for 5 min and separated on 10% pre-cast SDS–PAGE gels (Biorad). SDS-PAGE gels were analyzed by immunoblotting using rabbit anti-androgen receptor (AR) (1:400 dilution; Santa-Cruz, N-20) and rabbit anti-TRPM8 (1:400 dilution; Abcam, ab109308) antibodies. Each experiment was repeated at least three times. TRPM8 antibody specificity was validated using siRNA against the TRPM8 channel (Fig. S4D) as described previously^{24 (link)}.