Ab47285

The cells were lysed on ice in radio immunoprecipitation assay lysis buffer containing a mixture of proteinase and phosphatase inhibitors for 30 min and then centrifuged at 13,000 g for 10 min. The protein concentration was subsequently measured using a bicinchoninic acid kit. A total of 20 μg protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. Subsequently, the membranes were blocked with 5% BSA and incubated with the diluted primary antibodies against pFOXO3^S253 (1:2000, ab47285, Abcam), PI3K (1:5000, ab1549, Abcam), pPI3K^Y607 (1:5000, ab182654, Abcam), AKT1 (1:5000, ab235958, Abcam), pAKT1^S473 (1:50, ab812, Abcam) and Snail (1:50, ab53519, Abcam) at 4°C overnight. After 3 rinses with Tris-buffered saline Tween-20 (TBST), the membranes were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000, ab7090, Abcam) for 1 h. After 3 rinses with TBST, the membranes were visualized using a SuperSignal West Pico Kit (Thermo Fisher Scientific Inc). Images were captured using the ChemiDoc™ XRS (Bio-Rad, Inc., Hercules, CA, USA). β-actin was regarded as the loading control. The expression of target protein = gray value of target protein bands/gray value of β-actin.

Cell suspension was prepared and the cells were treated according to the preset drug concentration and time. Sodium dodecyl sulfate lysis buffer was added, and the cells were placed on ice for 20 minutes. Then, the protein concentration was measured using the bicinchoninic acid assay method, the sample was loaded and transferred to the membrane after electrophoresis, the milk blocking solution was sealed for approximately 1 hour, the primary antibodies were incubated, and the protein was then placed in a 4°C refrigerator (12–18 hours). The primary antibodies used were anti-Bcl-2-associated X protein (Bax) (ab32503, 1/1,000), anti-cleaved-caspase-3 (ab32042, 1/500), anti-B-cell lymphoma-2 (ab32124, 1/1,000), anti-AKT (ab233755, 1/1,000), anti-p-AKT (phospho T308; ab38449, 1/1,000), anti-FOXO3 (ab23683, 1 µg/mL), anti-p-FOXO3 (phospho S253; ab47285, 1/1,000), and anti-glyceraldehyde 3-phosphate dehydrogenase (all from Abcam, Boston MA, USA). The secondary antibody was added and incubated at room temperature in a shaker for 1 hour. The i-Bright FL1000 gel imaging system was then used for development and fixing.

For the immunofluorescence assay, podocytes and rat kidneys were fixed in 4% paraformaldehyde. The kidneys were cut into 5 μm sections. The cryosections were deparaffinized with xylene and rehydrated using a graded ethanol series. The podocyte cells and kidney sections were blocked with 1% bovine serum albumin for 1 h, incubated with primary antibodies for 1 h at room temperature in a humidified chamber, and then incubated with a donkey anti-goat 488-conjugated secondary antibody (ab150129, Abcam) or goat anti-rabbit 568-conjugated secondary antibody (ab175471, Abcam) at room temperature for 1 h. The following antibodies were used: rabbit anti-GLCCI1 (1:100, ab107491, Abcam), goat anti-nephrin (1:100, sc-19000, Santa Cruz Biotechnology), rabbit anti-podocin (1:100, sc-21009, Santa Cruz Biotechnology), goat anti-synaptopodin (1:100, sc-21536, Santa Cruz Biotechnology), rabbit anti-SGK1 (1:100, ab59337, Abcam) and rabbit anti-FOXO3A (1:100, ab47285, Abcam). All images were obtained using a Carl-Zeiss LSM 700 confocal microscope (Carl-Zeiss, Jena, Germany).