Vimentin ab8978

The cells were washed twice with phosphate-buffered saline (PBS) (GIBCO, Carlsbad, CA, USA) and lysed in RIPA lysis buffer (Sigma, St. Louis, MO, USA). Protein concentrations were determined using the Protein Assay Reagent (Bio Rad Laboratories, Hercules, CA, USA). Protein samples (30 μg) were loaded on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio Rad Laboratories, Inc.). Blocking was performed with 5% nonfat milk in a 1X mixture of Tris-buffered saline and Tween 20 (TBST). The Membranes were incubated overnight at 4°C with the following antibodies (at 1∶1000 dilutions in TBST with 5% non-fat milk): ADAM9 (#2099, Cell Signaling, Danvers, MA), CDH1 (E-cadherin, #610404, BD Science, Clontech, Palo Alto, CA, USA), CDH2 (N-Cadherin, #610921, BD Science, Clontech, Palo Alto, CA, USA), VIM (Vimentin, ab8978, Abcam, Cambridge, MA), ACTB (β-actin, ab8226, Abcam, Cambridge, MA) and EF1A (EF1α, #05-235, Millipore, Billerica, MA, USA). After washing and incubation with secondary antibodies (at 1∶2500 dilutions in TBST with 5% non-fat milk) for one h at room temperature, blotted proteins were detected using an enhanced chemiluminescence (ECL) system (Millipore, Billerica, MA, USA) with the BioSpectrum Imaging System (UVP, Upland, CA, USA).

Anti-LC3 (#12741), mTOR (#2983), phospho-mTOR (Ser2448) (#5536), PARP (#9542), and Caspase-3 (#9665) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-Beclin-1 (ab62472), ATG9A (ab108338), Ki-67 (ab15580), and Vimentin (ab8978) antibodies were from Abcam (Cambridge, UK). Anti-β-Actin (D110001) antibody was from Sangon Biotech (Shanghai, China). Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 555-conjugated goat anti-rabbit IgG were manufactured by Jackson ImmunoResearch (West Grove, PA, USA). DHT was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in ethanol. RAPA, CQ, and finasteride were from Selleck (Shanghai, China). RAPA and finasteride were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich); CQ was dissolved in ultrapure water.