Ab17026

Total protein was extracted from the treated A549 and H1299 cells using RIPA Lysis Buffer (Vazyme, cat. no. FD008). And the concentration was then quantitated using Pierce BCA protein assay kit (Rockford). The equivalent proteins in each group were isolated using 10% SDS-PAGE according to their molecular weights, and the separate proteins were transferred to PVDF membrane (Millipore). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight, followed by second antibody (Abcam) for 1 h. Finally, the membranes were treated with Enhanced ECL luminescence detection kit (Vazyme; E411-04), and the results were examined on FluorChem™ M System. The primary antibody included anti-FOXO3 (Abcam, ab17026), anti-FOXO1 (Abcam, ab39670), anti-GRB2 (Abcam, ab111031), and anti-GAPDH (Abcam, ab37168).

The total proteins were prepared, and their concentrations were determined using a Total Protein Extraction Kit (Solarbio, Beijing, China). Cell lysates were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, USA). Then, the membranes were blocked with 5% non-fat milk in TBST buffer. The following primary antibodies were used in the study: anti-AUF1 (1:1000, Abcam, ab61193), anti-FOXO3 (1:1000, Abcam, ab17026), anti-E-cadherin (1:1000, Abcam, ab1416), anti-Collagen 1 (1:1000, Abcam, ab6308), anti-α-SMA (1:1000, Abcam, ab5694), and anti-β-actin (1:1000, Abcam, ab8226). Next, the membranes were incubated with HRP-conjugated secondary goat anti-mouse (1:5000, Proteintech, Rosemont, IL, SA00001-1) or goat anti-rabbit (1:5000, Proteintech, SA00001-2) antibodies for 2 h at room temperature. The relative grey values of immunoreactive bands were calculated relative to those of β-actin levels.