Cells were washed twice with PBS and homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime Biotech, Suzhou, China) containing protease inhibitors at 4°C and centrifuged at 12,000g for 15 minutes. Then, 30 µg of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were blocked with 5% skim milk for 2 hours at room temperature in Tris-buffered saline. The membranes were then incubated overnight at 4°C with primary antibodies against PXN (Bioswamp, Wuhan, China): (molecular weight 102 kD PAB36563, 1:1000); hexokinase (HK)1 (120 kD, PAB30519, 1:1000); HK2 (102 kD, PAB30271, 1:1000); glucose transporter 1 (GLUT1; 54 kD, PAB34949, 1:1000), PI3K (124 kD, PAB30084, 1:1000), phospho-PI3K (84 kD, phosphorylation sites Y467/Y199/Y464, PAB4641-P, 1:1000), AKT (60 kD, PAB30596, 1:1000), phospho-AKT (56 kD, phosphorylation site S473, PAB4318-P, 1:1000), mTOR (290 kD, PAB30674, 1:1000), and phospho-mTOR (289 kD, phosphorylation site S2448, PAB36313-P, 1:1000). Glyceraldehyde-3-phosphate dehydrogenase (36 kD, PAB36269, 1:1000; Bioswamp) was selected as the internal reference.
The membranes were washed with Tris-buffered saline and incubated in biotinylated goat immunoglobulin G secondary antibody (SAB43714, 1:20000; Bioswamp) for 2 hours at room temperature. Immunoreactivity was visualized by colorimetric reaction using an enhanced chemiluminescence substrate buffer (MilliporeSigma). Membranes were scanned with a Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, USA).