Ab195039

Kidney tissue was added to RIPA lysate (Thermo Fisher Scientific, United States) containing phosphatase inhibitor and protease, followed by being disrupted by an ultrasound. Then, the sample was centrifuged at 14,000 g at 4°C for 15 min, and the supernatant was collected. The BCA protein detection kit (Pierce, United States) was utilized for measuring the protein concentration. 20 μg protein was loaded. After 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the product was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The membrane was blocked with 5% bovine serum albumin at 37°C for 2 h, and was incubated with primary antibodies overnight at 4°C. Antibodies included EIF4B (1/10000; ab134138; Abcam, United States), RICTOR (1/1000; ab219950; Abcam, United States), PRKCB (1/2000; ab195039; Abcam, United States), and GAPDH (1/10000; ab8245; Abcam, United States). After being washed 3 times with TBST, the membrane was incubated with HRP-labeled secondary antibody (1/2000; ab7097; Abcam, United States) for 2 h at 37°C. After being washed three times, the protein expression was detected by a gel quantitative analysis system following treatment with ECL (Solarbio, Beijing, China) with GAPDH as the reference control.