Ab140307

Total proteins from cells and tissues were extracted by the RIPA lysate. Protein quantification was conducted with a BCA protein quantification kit. An appropriate amount of protein sample was denatured at 100°C for 3–5 min and then subjected to protein electrophoresis using 10% SDS-PAGE for 1 h. the samples were transferred to PVDF membranes. The membranes were blocked in Tris Buffered Saline with Tween 20 blocking solution containing 5% skim milk powder at room temperature for 2 h. The membranes were washed three times with TBST and incubated overnight at 4°C using primary antibodies against MMP-2, MMP-9, p-PI3K, PI3K, p-AKT, AKT, and GAPDH (ab92536, ab76003, ab278545, ab140307, ab192623, ab8805, ab8245; all from Abcam, Shanghai, China). The next day, the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (ab96899) and goat anti-mouse secondary antibody IgG (ab96879) dilution buffer were added, and membranes were incubated with these secondary antibodies at room temperature for 2 h. The enhanced chemiluminescence reagent was added dropwise, and the membranes were imaged by the gel imaging system. The gray value of each band was analyzed by the ImageJ software. The results were expressed as the ratio of the gray value of the target protein to GAPDH, the internal reference protein.

The anti-p-PI3K antibody (ab182651), anti-PI3K antibody (ab140307), anti-AKT antibody (ab8805), anti-p-AKT antibody (ab38449), anti-LC3 antibody (ab192890) and anti-GAPDH antibody (ab8245) were purchased from Abcam. Antibodies against caspase 3 (19677-1-AP), caspase 7 (27155-1-AP), caspase 9 (10380-1-AP), FOXO1 (66457-1-Ig), FOXO3 (66428-1-Ig), and ATIC (10726-1-AP) and secondary antibodies (SA00001-1 and SA00001-2) were purchased from Proteintech (Wuhan, China). Cell samples were lysed in sodium dodecyl sulfate (SDS, Beyotime, Shanghai, China). The protein concentration was determined with the Enhanced BCA Protein Assay Kit (Beyotime), and proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1 hour, membranes were incubated with a specific primary antibody overnight at 4 °C and then incubated with the secondary antibody at room temperature for 2 hours. Protein bands were detected using an enhanced chemiluminescence (ECL) system (Pierce Biotech, Rockford, Illinois, USA). An anti-GAPDH antibody (1:1,000, Sigma, St. Louis, Missouri, USA) was used to detect GAPDH as a loading control.

After cell treatment, the medium was discarded. Supplementation of protein lysis solution (Roche) was completed for separating the total protein. Then, 50 μg of total protein was sampled on 12% polyacrylamide gel for 2-hour electrophoresis at 100 V. Afterward, the electrophoresed total protein was transferred to polyvinylidene fluoride (PVDF) membranes. One hour after being sealed with 5% skimmed milk at RT, the membranes were flushed three times (10 minutes each time) with Tris-buffered saline with Tween-20 (TBST) for overnight incubation at 4°C with the following primary antibodies (Abcam, Cambridge, UK, concentration 1:1000): Anti-Bax antibody (ab182734), Anti-Bcl-2 antibody (ab692), Anti-Cleaved Caspase-3 antibody (ab214430), Anti-p-AKT antibody (ab38449), Anti-AKT antibody (ab8805), Anti-p-PI3K antibody (ab278545), Anti-PI3K antibody (ab140307), and Anti-HIF-1 alpha Antibody (ab179483). Next, the membranes were maintained with the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG (ab6205718) (concentration 1:2500) at RT for one hour after being rinsed with TBST. Following 3 washes with TBST (10 minutes/time), the membranes were exposed with enhanced chemiluminescence (ECL) chromogenic agent (Millipore, Bedford, MA, USA), and a membrane scanner was utilized for imaging [24 (link)].