Tbs 0.1 tween 20

Cells were lysed with SDS lysis buffer (2% SDS, 10% Glycerol, 50mM Tris pH 6.8) and boiled at 95°C for 30min. Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFischer) following manufacturer’s recommendations. Samples were ran in NuPAGETM 10% Bis-Tris Midi electrophoresis gels (Invitrogen) using NuPAGETM MES SDS running buffer (ThermoFischer Scientific). Proteins were subsequently transferred to an Immobilon-P polyvinylidene Difluoride (PVDF) membrane (Merck Millipore) using the wet-transfer method (Bio-Rad) in a transfer buffer consisting of 20% (v/v) methanol and 10% protein electrophoresis buffer (24.77 mM Tris and 0.192 M glycine).
For western blotting membranes were blocked in a solution of 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) dissolved in TBS-0.1% Tween 20 (Sigma-Aldrich) then incubated at 4°C overnight with the primary antibodies diluted in 5% (w/v) BSA/TBS-T. The membranes where then washed and incubated with anti-mouse and/or anti-rabbit secondary antibodies (LI-COR Biosciences) for 2h at RT diluted in 5% (w/v) BSA/TBS-T then washed and imaged using the LI-COR Odyssey (LI-COR Biosciences). Images were analyzed using Image Studio Lite software (LI-COR Biosciences).

Twenty microlitres of each of 8 replicates of DMSO/control-treated lysate from the luciferase assay were pooled and the total amount of protein was assayed by bicinchoninic acid assay (BCA) (Pierce™, Appleton, WI, USA). Equal amounts of protein samples were loaded on a 10% SDS-PAGE gel and electrophoresed at 120 V for 90 min. Proteins were transferred to PVDF membrane (Millipore, Burlington, MA, USA) at 25 V for 15 min using a TransBlot^® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was then blocked for 30 min at room temperature with 5% non-fat skim milk in TBS, followed by incubation with 1/1000 dilution of AR (441) mouse monoclonal antibody (sc-7305, Santa Cruz Biotechnologies, Dallas, TX, USA) and GAPDH Antibody (G-9) HRP mouse monoclonal antibody (sc-365062, Santa Cruz Biotechnologies) overnight at 4 °C. Membranes were then washed and incubated with 1/1000 dilution of Donkey Anti-Mouse IgG Polyclonal Antibody (IRDye^® 680RD; 925-68072; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature, washed 3 times with TBS 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) and bands visualized using Odyssey Li-Cor Scanner.