As061

Cells were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), and protein levels were quantified using a BCA protein assay kit (P0012S, Beyotime). The extracted cellular proteins were mixed with loading buffer (v/v = 4:1) (P1040, Solarbio) and boiled for 10 min. Protein samples (30 μg per well) were separated by 8% SDS–PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with 5% BSA in 1 × TBST (0.2% Tween-20, pH = 7.6) buffer for 2 h and then incubated with primary antibodies against GAPDH (1:100,000, A19056, ABclonal, Wuhan, China) and ARID5B (1:2000, NBP1-83622, Novus, Shanghai, China) at 4 °C overnight. After being washed with 1 × TBST buffer four times (10 min each time), the membranes were then incubated with HRP-conjugated mouse anti-rabbit (1:5000, AS061, ABclonal) and goat anti-mouse (1:5000, AS003, ABclonal) secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using an ECL reagent (PE0010, Solarbio) and photographed by a Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Image-Pro Plus (v 6.0) software was used to perform the densitometric semiquantitative analysis of protein bands. The protein expression was normalized against GAPDH.

Monoclonal anti-Actin Beta (ACTB) antibody (AC026, ABclonal Technology, Wuhan, China), monoclonal anti-GFP antibody (AE012, ABclonal Technology), monoclonal anti-RFP antibody (AE020, ABclonal Technology), monoclonal anti-His antibody (AE003, ABclonal Technology), polyclonal anti-phospho-AKT antibodies (AP0140, ABclonal Technology), polyclonal anti-phospho-FOXO antibodies (AP0684, ABclonal Technology), mouse control IgG (AC011, ABclonal Technology), rabbit control IgG (AC005, ABclonal Technology), Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG light chain (AS061, ABclonal Technology), polyclonal anti-AKT antibodies (WL0003b, Wanleibio, Shenyang, China), polyclonal anti-FOXO antibodies (WL02891, Wanleibio), Alkaline phosphatase (AP)-conjugated horse anti-mouse IgG (ZB2310, ZSGB-BIO, Beijing, China), and AP-conjugated goat anti-rabbit IgG (ZB2308, ZSGB-BIO).