Enhanced chemiluminescence

A549, H1299, or H1975 cells were collected and washed with phosphate-buffered saline (PBS) three times, lysed with standard RIPA buffer (Beyotime, China) on ice for 30 mins, and centrifuged at 13,000×g for 10 mins at 4°C. The supernatants were collected as detecting samples. The protein concentration was determined using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Aliquot proteins of the samples were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, separated by electrophoresis, and transferred onto a poly(vinylidene fluoride) membrane (Millipore, Bedford, MA, USA). After reaction with the corresponding primary and secondary antibodies, the blotted membranes were visualized using enhanced chemiluminescence (Meilun, Dalian, China) reagents.

The proteins in the renal tissues were extracted with lysis buffer. After denaturation, 40 μg of protein was loaded on the 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. After blocking overnight at 4 °C with 4% skimmed milk, the membranes were incubated with the anti-GAPDH (Multi Sciences, Hangzhou, China) and anti-PTEN (Multi Sciences, Hangzhou, China) antibodies. After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Multi Sciences, Hangzhou, China) to identify the respective proteins. The bands were visualized using enhanced chemiluminescence (Meilun, Shanghai, China) and quantified with Image J software. GAPDH was used as an internal control.