A11021

Cells were lysed in lysis buffer (#KGP250/KGP2100, Keygen Biotech, Nanjing, China), and proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) before transferring to PVDF membranes, according to the standard manufacture’s protocol. Vinculin (Gudiño et al. 2021 (link)) and β-Actin were used as internal loading control. The following primary antibodies were used in this study: galectin-3 (#ab2785, Abcam; dilution rates of 1:1000), foxc1 (#ab227977, Abcam; dilution rates of 1:1000), active caspase-3/caspase-3 (#A11021 and #A19654, ABclonal, Wuhan, China; dilution rates of 1:500), and vinculin (#26520-1-AP, Proteintech, China; dilution rates of 1:2000). All primary antibodies were incubated with PVDF membranes overnight at 4 °C. The incubation time for active caspase-3/caspase-3 primary antibody needed to be appropriately longer. Images of protein bands were captured using Syngene GeneGenius (SYNGENE, GeneGnome XRQ NPC, Cambridge, UK.).

For western blotting, kidney tissues or podocytes were extracted and quantified. The protein samples were then boiled at 95 °C for 10 min and separated on a 6–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then incubated with primary antibodies overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for protein expression visualization. Primary antibodies were used as follows: NPHS2 antibody (1:500, 20384-1-AP, Proteintech), phosphorylated Nephrin antibody (1:10000, ab80299, Abcam), Active Caspase-3 antibody (1:500, A11021, ABclonal), WT1 antibody (1:1000, A2446, ABclonal), β-actin antibody (1:3000, AB0035, Abways), Bax antibody (1:500, 380709, ZENBIO), RAC1 antibody (1:1000, ab155938, Abcam), β-tubulin antibody (1:3000, AB0039, Abways), OLR1 antibody (1:500, 11837-1-AP, Proteintech), SR-A1 antibody (1:500, 382017, Zenbio), CD36 antibody (1:500, 381350, Zenbio), IGF-1R antibody (1:50, sc-81464, Santa Cruz).