Ab32199

Cells were lysed in RIPA Lysis Buffer and a BCA Protein Assay kit (Thermo Fisher Scientific) was used to determine the protein concentration. Proteins of each sample were separated by SDS-PAGE gel. Then, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% non-fat milk, followed by the incubation with primary antibodies at 4°C overnight: anti-Bax (Abcam; ab32503) (1:1000), anti-active caspase 3 (Abcam; ab2302) (1:1000), anti-Bcl-2 (Abcam; ab32124) (1:1000), anti-β-actin (Abcam; ab8227) (1:1000), anti-PTEN (Abcam; ab32199) (1:1000), anti-p-PI3K p85 (p-p85, Abcam; ab182651) (1:1000), anti-p-Akt (Abcam; ab38449) (1:1000). The membranes were washed in TBST three times then incubated with secondary antibodies for 1 hr at room temperature. Chemiluminescence (Millipore Corporation, Billerica, MA, USA) were applied to measure protein expression using densitometry analysis (ImageJ software).

Protein lysates were collected from A549 or NCI-H1975 cells using RIPA Buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktail. Protein concentrations were estimated using bicinchoninic acid protein assay kit (CWBIO, Beijing, China) and proteins were denatured for 10min at 95°C. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with non-fat milk in Tris-buffered saline and Tween 20. After that, the membranes were incubated with primary antibodies against Bax (Abcam, ab32503, 1:5000), Bcl-2 (Abcam, ab32124, 1:1000), Cleaved caspase3 (Abcam, ab2302, 1:500), Caspase3 (Abcam, ab32150, 1:1000), PTEN (Abcam, ab32199, 1:10,000), p-PI3K (Abcam, ab182651, 1:1000), p-AKT (Abcam, ab38449, 1:1000) and GAPDH (Abcam, ab9485, 1:2000) at 4 °C overnight. On the second day, the membranes were incubated with the corresponding IgG-HRP secondary antibody (Abcam, ab205718, 1:20,000) at room temperature for 1 h. Signals were developed using ECL (Sigma-Aldrich, MO, USA) and analyzed by Image J software (NIH, USA).

Cells were washed in phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The protein concentration was evaluated using a bicinchoninic acid protein assay kit (Bioswamp, PAB180007, Wuhan, China). Equivalent amounts of proteins (30 μg) from each sample were subjected to sodium dodecyl-polyacrylamide electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked in 5% fat-free milk for 2 hours at room temperature, and incubated with the following primary antibodies: PTEN (ab32199, 1:10,000, abcam), PI3K (ab191606, 1:10,000, abcam), p-PI3K (ab182651, 1:10,000, abcam), mTOR (ab32028, 1:10,000, abcam), p-mTOR (ab109268, 1:10,000, abcam), GLUT1 (ab652, 1:10,000, abcam), LDHB (ab85319, 1:10,000, abcam), HK2 (ab209847, 1:10,000, abcam), PKM2 (ab150377, 1:10,000, abcam), or GAPDH (PAB36264, 1:10,000, Bioswamp). Then, the membranes were washed with Tris-buffered saline and incubated with goat anti-rabbit IgG secondary antibody (SAB43711, 1:10,000, Bioswamp) for 2 h at room temperature. An enhanced chemiluminescence kit (Pierce) was used to detect specific bands and autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA) using GAPDH as a control. For each group, the quantification was triplicated.