Ab108341

The cells were washed with ice-cold PBS three times and lysed with RIPA lysis buffer (Sangon Biotech, China) with a protease inhibitors cocktail (Roche, China). The total protein content in the supernatant was measured by the BCA protein assay kit (Sangon Biotech, China) according to the manufacturer’s instruction. The samples were boiled with SDS-PAGE loading buffer (Solarbio, China). Twenty micrograms of protein was added to each lane, separated in 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA) in a Trans-Blot apparatus (Tanon, China). 5% BSA diluted in TBS-T (0.05% Tween 20 in TBS) was used to block the membranes, and the primary antibodies with optimized concentrations, including AR (1:1000, ab108341, Abcam, USA), Actin (1:1000, D191048, Sangon Biotech, China), PSA (1:1000, 10679, Proteintech, China), were added to the membranes overnight at 4 °C. The membranes were incubated with the corresponding secondary antibodies conjugated with HRP (1:10000, D110011, Sangon Biotech, China) for 1 h at room temperature, and the bands were visualized by an enhanced chemiluminescence detection system.

Cells were lysed with RIPA buffer (Sigma-Aldrich) with added protease inhibitors (Sigma-Aldrich). Proteins were obtained by centrifugation at 12,500 rpm at 4 °C for 30 min and quantified using an EZQ Protein Quantitation Kit (Life Technologies) according to manufacturer’s instructions. Proteins (30 µg) were separated by electrophoresis on SDS-PAGE (Bio-Rad). SeeBlue Plus2 (Invirogen) was used for sizing and visualization of the gel running pattern and protein transfer. Resolved proteins were transferred on immobilon PVDF membranes (Millipore) at 20 V for 30 min. Membranes were air-dried, wet with methanol, washed with TBS-T and blocked at 4 °C overnight in 1% (w/v) non-fat skimmed milk/TBS-T. Membranes were incubated with antibodies against AR (1:500 ab108341), GABBR1 (1:500 ab75239) from ABCAM, GAPDH (1:20000 5174 s), ENO2 (1:200 9536 s) obtained from Cell Signaling and GRP (1:500 sc-271045) from Santa Cruz Biotechnology at 4 °C overnight.
Blots were then incubated with anti-rabbit secondary antibody (1:5000 ab99697) obtained from ABCAM or anti-mouse secondary antibody (1:300 7076 s) from Cell Signaling conjugated to horseradish peroxidase (HRP) at room temperature (RT) for 2 h. Chemiluminescence signals were detected using ChemiDoc XRS system (BioRad). Western immunoblots were quantified using ImageJ software.