Protease phosphatase inhibitor cocktail

The cell lysates were prepared with RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (MedChemExpress, Cat# HY-K0010) and 100 mol/L PMSF on ice. The relative protein concentration was measured using the BCA protein assay kit (Thermo-PIERCE, Cat# 23225). The protein lysates were separated using SDS-PAGE on 10% polyacrylamide gels, and molecular weight standards were determined with a prestained protein marker. The separated proteins were electro-transferred onto nitrocellulose filter (NC) membranes at ice-cold temperatures, followed by blocking in 5% bovine serum albumin for 1 h at room temperature. The appropriate primary antibodies, including anti-LOXL2 (Abcam Cat# ab96233, 1:1 000 dilution), anti-HIF-1 alpha (Abcam Cat# ab179483, 1:500 dilution), and anti-GAPDH (Abcam Cat# ab9485, 1:10 000 dilution), were rotated with the membrane at 4 °C overnight. Fluorescent secondary antibodies (LI-COR Biosciences, Cat# 926-32211, 1:10 000 dilution) were added for 2 h. Images were obtained using the LI-COR Odyssey CLx system, and densitometric analysis of protein bands was conducted using ImageJ.

Using RIPA lysis buffer and protease/phosphatase inhibitor cocktail (MedChemExpress, NJ, USA) with 1 mM phenylmethyl sulphonyl fluoride PMSF (Beyotime Biotech, Shanghai, China), the whole protein in each cell sample was extracted and quantified with a BCA protein assay (Beyotime Biotech, Shanghai, China). After blocking the protein with the primary antibodies against c-Myc, SOX2, p-Akt (Ser473), Akt, MCL-1, Bcl-2, FAS, and cleaved-caspase 3 beta-actin (1 : 1000, Cell Signaling Technology, Danvers, MA USA), the incubation period was extended overnight at 4°C. Secondary antibody conjugated to HRP (Beyotime Biotech, Shanghai, China, 1 : 4000) was incubated for 2 hours at room temperature with protein. The eECL Western blot kit (Beyotime Biotech, Shanghai, China) was utilized to detect the protein.