Ab109308

Cells were seeded in Petri dishes with the appropriate medium and grown to a confluency of 80%. Before cell lysis, Petri dishes were kept on ice and the cells were washed twice with ice-cold PBS. Cells were lysated in RIPA Buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with the following protease inhibitors: 2 mg/ml aprotinin, 1 mM Na orthovanadate, 0.1 mM PMSF and 10 mM Sodium Fluoride. Lysates were centrifuged at 4 °C for 10 min at 12,000 × g. Protein concentrations were determined using a Bicinchoninic Acid Kit (BCA kit, Sigma). 50 µg of sample were resuspended in SDS sample buffer, heated at 95 °C for 5 min and separated on 10% pre-cast SDS gel (Biorad). Polyvinylidene fluoride membranes were properly blocked and then incubated overnight with rabbit anti-TRPM8 (1:400 dilution; Abcam, ab109308) and with rabbit anti-AR (1:400 dilution; Santa-Cruz, N-20). The membrane was then washed with TBS containing 0.1% Tween 20 and incubated with the appropriate horseradish-peroxidase-conjugated antibodies (SantaCruz). Chemiluminescence assays were conducted using the SuperSignal West Dura chemiluminescent substrate (Thermo Fischer Scientific).

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)}.

The biotinylation assay was performed using the NHS-LC-LC-biotin kit (Pierce, Etten-Leur, The Netherlands) after homogenizing the cells in 600 µl of lysis buffer, as described previously^{24 (link)}. Biotinylated proteins were precipitated using neutravidin-agarose beads (Pierce). TRPM8 and AR expression was analyzed by immunoblotting of the precipitates (PM fraction) or of total cell lysates using the anti-TRPM8 (1:400 dilution; Abcam, ab109308) and the anti-AR antibodies (1:400 dilution; Santa-Cruz, N-20). Each experiment was repeated at least three times.