Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g secondary antibody

At 24 h post-transfection, C666-1 and 13-9B cells were harvested, and 1×10⁵ cells were mixed with 1 ml RIPA solution (Sangon Biotech Co., Ltd.) to extract total proteins. The BCA method (Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Proteins were incubated with sample buffer and boiled for 5 min to denature the proteins. Protein samples (30 µg per lane) were subsequently loaded on a 12% gel, resolved using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with PBS containing 5% skimmed milk at 22°C for 2 h. Membranes were incubated with rabbit polyclonal primary antibodies against PTEN (1:1,200; cat. no. ab31392; Abcam) or GAPDH antibody (1:900; cat. no. ab9485; Abcam) at 4°C overnight, and subsequently with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,200; cat. no. MBS435036; MyBioSource, Inc.) at 22°C for 2 h. Enhanced chemiluminescence reagent (Sigma-Aldrich; Merck KGaA) was used to detect the signal on the membrane. Densitometry analysis was performed using Image J version 1.46 (National Institutes of Health). GAPDH was used as the loading control.

Total protein was extracted from cultured cells using RIPA Lysis and Extraction Buffer (Invitrogen, Carlsbad, CA, USA). The concentration of total protein was detected using the BCA Protein Assay Kit (Beyotime; Shanghai, China). Equal amounts of protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk for 2 h, the membranes were incubated overnight with specific primary antibodies targeting FOXO3 (cat. No. ab109629; Abcam, Cambridge, UK) or GAPDH (cat. No. ab181603; Abcam) and then probed with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. No. ab205718; Abcam). Finally, the Enhanced Chemiluminescence Detection System (Pierce; Thermo Fisher Scientific, Inc.) was used to develop protein signals.

Protein samples from 16HBE cells were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors and phosphatase inhibitor (Beyotime) at 4°C for 30 min. Then, the concentration of protein was calculated using the BCA Protein Assay kit (Beyotime). Protein samples were subjected to 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene membrane. Subsequently, the membranes were blocked with 5% skimmed milk. The primary antibodies against E-cadherin, N-cadherin, Vimentin, PI3K, p-AKT, t-AKT, PARP-1 (CST, Boston, MA, USA), Snail, ZEB1 (ABclonal, Wuhan, China), and GAPDH (Abcam, Cambridge, UK) were incubated with the membranes at 4°C overnight. Then the membranes were washed three times and incubated for 2 h with horseradish peroxidase-conjugated goat antirabbit Immunoglobulin G secondary antibody at room temperature (Abcam). Finally, the protein on the membrane was visualized with the Chemiluminescent ECL Detection System (Tanon, Shanghai, China) and quantitated by ImageJ version 1.48 (National Institutes of Health, Bethesda, MD, USA).