Anti aldh1a1

Protein extracts from cells and tissues were quantified. Thirty microgram total protein were separated on a 10% SDS-PAGE gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with BSA and incubated at 4°C overnight with primary antibodies anti-ADH1 (CST, Framingham, MA, USA, 1:500), anti-NR5A1 (CST, 1:1,000), anti-HSD3B (Biorbyt, Cambridge, UK, 1:200), anti-StAR (CST, Framingham, MA, USA, 1:1,000), and anti-ALDH1A1 (Protein Tech, Wuhan, China, 1:500). The membranes were washed with tris-buffered saline (TBS buffer), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1.5 h. The membranes were then washed with TBS buffer three to five times and visualized by enhanced chemiluminescence (ECL) detection. The protein expression was normalized to the GAPDH levels (27 (link)).

The experiment was carried out following our previously published protocol [13 (link)]. Anti-FOXD1 antibody was purchased from Genetex (1:1000; CA, USA). Anti-E-cadherin, anti-N-cadherin, and anti-vimentin antibodies were purchased from Cell Signaling Technology (1:1000; MA, USA); anti-CD44, anti-ALDH1A1, anti-BMI1, and anti-SNAI2 antibodies were purchased from Proteintech (1:1000; Wuhan, China). The gray value of the band was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).