Western blot analysis

The kidney tissues and cells in each group were lysed, and the total protein was extracted. Equal amounts of protein (40 μg) were electrophoresed on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to PVDF, blocked and then incubated with the following primary antibodies against the following antigens at 4 °C overnight: PI3K, p-PI3K, AKT, p-AKT, E-cad, α-SMA (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Bioworld Technology, Inc., Atlanta, GA, USA). The filter membrane was washed with PBS buffer three times and incubated with a horseradish peroxidase (HRP) –labeled secondary antibody for 1 h at room temperature. With GAPDH expression as a reference, the relative protein expression was described as the ratio of the staining intensity of the target band to the staining intensity of the reference band.