Anti aldh1

The experimental procedure for Western blotting analysis that was used in this study was followed as described in our previous study [29 (link)]. The horseradish peroxidase-conjugated IgG that was anti-mouse and anti-rabbit (Thermo Fisher Scientific, New York, NY, USA) was used in this study. GAPDH was used as the control and for quantification. Antibodies purchased from Santa Cruz, USA: anti-EPCAM (1:1000, sc-25308), anti-ALDH1 (1:300, sc-374149), anti-ALDH2 (1:300, sc-100496), anti-KLF4 (1:1000, sc-166100), anti-GAPDH (1:1000, sc-47724). Antibodies purchased from Cell Signaling Technology, USA: anti-SNAI2 (#9585, 1:1000), anti-SOX2 (#3579, 1:1000), anti-OCT4 (#2840, 1:1000), anti-NANOG (#4903, 1:1000), anti-β-catenin (#8480, 1:1000), anti-c-Myc (#18583, 1:1500 dilution). Anti-PCNA (#60097-1-Ig, 1:5000) were purchased from Proteintech. The signal intensity was quantified using the protein imprinting imaging system (Tanon 5200, Better Tanon, Shanghai, China).

Enzalutamide (Cat# A10562) was purchased from Adooq Bioscience, Irvine, CA, USA, and all other reagents purchased were of GLP grade. Antibodies including Anti-AR (Cat# 5153), Anti-AR-v7 (Cat# 19672), anti-POU5F1 (OCT4) (Cat# 2750S) were purchased from Cell Signaling Technologies, Beverly, MA, USA. The anti-ALDH1 (Cat# SC-166362), anti-SOX2 (Cat# SC-365823), anti-α-GAPDH (Cat# SC-47724), goat anti-mouse IgG-HRP (Cat# SC-2005), bovine anti-goat IgG-HRP (Cat# SC-2350), and goat anti-rabbit IgG-HRP (Cat# SC-2004) antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

The cells were harvested with RIPA buffer. The protein samples (30–50 μg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The blots were blocked for 1 h, and then incubated with the primary antibodies at 4 °C overnight. The blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). The protein was visualized with a Pierce ECL Western Blotting Substrate detection system (Thermo Fisher Scientific, Bremen, Germany). β-Actin was used as the loading control. The primary antibodies used in this study included anti-FOXM1, anti-FOXO3a, anti-p53, anti-ABCA2, anti-SOX2, anti-ALDH1, anti-GAPDH and anti-β-actin purchased from Santa Cruz (Santa Cruz, CA, USA); anti-FOXO1, anti-FOXC2 and anti-ABCC5 purchased from Abcam (Shanghai, China); and anti-E-cadherin, anti-vimentin, anti-Snail, anti-ZEB-1, anti-p38, anti-p44/42, anti-AKT, anti-AKT473 and anti-AKT308 purchased from Cell Signaling Technology (CST, Danvers, MA, USA).