Hrp conjugated anti mouse or anti rabbit secondary antibodies

The p-GSK-3β inhibitor TWS-119 was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The p-Akt inhibitor LY294002 was purchased from Cell Signaling Technology (Danvers, MA, USA). For miRNA experiments, all miRNAs were obtained from Dharmacon (Lafayette, CO, USA). Antibodies used in the experiments included anti-Axl and cyclin D1, both obtained from Santa Cruz Biotechnology (Dallas, TX, USA), as well as p-Axl (Tyr702), Akt, p-Akt (Ser473), GSK-3β, p-GSK-3β (Ser9), and GAPDH, all sourced from Cell Signaling Technology. The β-catenin antibody was obtained from BD Biosciences (San Jose, CA, USA), and the cellular Myc protein (c-Myc) antibody was sourced from Abcam (Cambridge, UK). The α-tubulin antibody came from Proteintech (Wuhan, China). Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), and Alexa Fluor 555 anti-mouse immunoglobulin G (IgG) was obtained from Invitrogen (Carlsbad, CA, USA).

Cells were washed with ice-cold PBS twice and lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) that contained protease inhibitors (Thermo Fisher Scientific) and, where necessary, phosphatase inhibitors (Phospho STOP tablets; Roche). Cell debris was removed by centrifugation at 14,000 g for 15 min at 4°C. Protein concentrations were determined by the Bradford protein assay (Bio-Rad). Equal amounts of proteins were mixed with SDS sample buffer, boiled at 95°C for 5 min, and separated by SDS-PAGE. The resolved proteins were then transferred on polyvinylidene difluoride (PVDF) membranes (0.45 µm Hybond P; GE Healthcare Life Science). Blots were blocked for 1 h at RT with 5% nonfat milk in TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies in blocking solution. Membranes were washed three times with TBS-T and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies for 1 h at RT (Jackson ImmunoResearch). After washing three times with TBS-T, proteins were visualized with ECL Western blotting substrate.