Anti mek1

Cells, untransfected or transfected with siRNA, were lysed in 70 μL of lysis buffer (50 mM Tris HCl pH 7.5, 0.1% Triton, 5 mM EDTA complemented by Proteases and Phosphatases inhibitors (Chemicon Millipore, Billerica, MA, USA and Sigma Aldrich, Saint-Louis, MO, USA) at 1/100). Western blots were performed as previously described [44 (link),45 (link)] using monoclonal rabbit antibodies, anti-Int6 (1/1000, Abcam, Paris, France), anti-HIF-2α (1/1000, Novus Biologicals, Cambridge, UK), anti-HIF-1α (1/1000, Cayman Chemical, Ann Harbor, MI, USA), anti-Caspase 3/7 (1/1000, Cell Signaling, Danvers, MA, USA), anti-cleaved caspase 3/7 (1/1000, Cell Signaling, Danvers, MA, USA) anti-PARP (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bax (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bcl-XL (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bcl-2 (1/1000, Cell Signaling, Danvers, MA, USA), anti-MEK1 (1/1000, Cell Signaling, Danvers, MA, USA), anti-ERK (1/1000, Cell Signaling, Danvers, MA, USA), anti-phospho ERK (1/1000, Cell Signaling, Danvers, MA, USA), and were normalized using a mouse monoclonal antibody anti-actin (1/10,000, Chemicon Millipore, Billerica, MA, USA) or a rabbit polyclonal antibody anti-β-tubulin (1/1000, Cell Signaling, Danvers, MA, USA). Gel quantification was performed using ImageJ (Windows 1.47, Research Services Branch, NIH, Bethesda, MD, USA).

Tumor cells were lysed with RIPA Lysis Buffer and Phenylmethanesulfonylfluoride (PMSF, Beyotime Institute of Biotechnology, China), followed by denaturation at 100° C. The BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China) was applied to determine the protein concentrations of samples. An equal amount of protein was loaded, separated by 10% SDS-PAGE gel, and blotted onto a nitrocellulose membrane. Then, the membrane was trimmed and blocked by 5% skim milk at room temperature for 2 h and incubated with primary antibodies at 4° C overnight. The antibodies include anti-AKR1C3 (ab236656, Abcam, Cambridge, MA, USA), anti-AKR1D1 (ab254943, Abcam, Cambridge, MA, USA), anti-MEK1 (12671, Cell Signaling Technology Inc, CST, MA, USA), anti-p-MEK1 (9127, Cell Signaling Technology Inc, CST, MA, USA), anti-p-Erk1/2 (4370, Cell Signaling Technology Inc, CST, MA, USA), anti-AR (19672, Cell Signaling Technology Inc, CST, MA, USA), anti-ID1 (ab168256, Abcam, Cambridge, MA, USA), and anti-GAPDH (5174, Cell Signaling Technology Inc, CST, MA, USA). Then the membranes were washed with TBST solution four times and incubated with the HRP-conjugated secondary antibody for 1 h at temperature. The protein band was finally incubated with enhanced chemiluminescence and exposed to an X-ray film.