Anti synaptopodin

The collected kidney tissues and podocytes from diverse groups were lysed with RIPA Buffer on ice for 20 min at 4°C to obtain the total protein. After determining the concentration of total protein, equivalent amounts of protein were separated on 10% SDS-PAGE gels and subsequently transferred onto PVDF membranes. Membranes were blocked with skimmed milk and then incubated with primary antibodies as followes:
anti-SIRT1 (#AF0282, 1:1000; Beyotime), anti-β-actin (#AF0003, 1:1000; Beyotime), anti-Desmin (#AF1414, 1:2000; Beyotime), anti-Nephrin (#PA5-106921, 1:2000;
Thermo Fisher Scientific, Inc.), anti-Synaptopodin (#PA5-21062, 1:2000; Thermo Fisher Scientific, Inc.), anti-P-cadherin (#13-2000Z, 1:1000; Thermo Fisher Scientific, Inc.), and anti-N-cadherin (#AF0243, 1:800; Beyotime). Then, the membranes were rinsed thrice before incubation with HRP-labeled Goat Anti-Rat IgG secondary antibody.
Finally, by using an ECL kit (Beyotime), the protein bands were visualized, of which intensities were measured by Image J 6.0 software..

After finishing diverse treatments, podocytes were fixed with 2% formaldehyde, followed by permeabilized with 0.3% Triton-X 100 and blocked with PBSB solution.
Then, podocytes were incubated with anti-Nephrin (#PA5-106921, Thermo Fisher Scientific, Inc.) or anti-Synaptopodin (#PA5-21062, Thermo Fisher Scientific, Inc.) antibodies for 1 h. Next, cells were washed thrice with cold PBS and then incubated with Cy3 conjugated anti-rabbit secondary antibodies (#A0516, Beyotime, Jiangsu, China) for 45 min. Coverslips were washed and mounted on DAPI Fluoromount-G. Finally, staining podocytes were observed and imaged under the Zeiss confocal microscope.