Immunofluorescence staining of cultured podocytes

The podocyte samples were fixed with 4% paraformaldehyde for 20 min at room temperature, rinsed with PBS (cat. no. KGB5001; Nanjing KeyGen Biotech Co., Ltd.) and then permeabilized with 0.5% Triton X-100 (Applygen Technologies, Inc.) in PBS for 30 min at room temperature. Non-specific binding was blocked with 5% BSA (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. The podocytes were incubated with primary antibodies overnight at 4˚C. The following day, the podocytes were washed with PBS, labeled with secondary antibodies using 2 drops/ml for 30 min at room temperature and incubated with Alexa Fluor 488 Phalloidin (cat. no. A12379 Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. Finally, podocytes were washed with PBS and sealed using antifade mounting reagent containing DAPI (cat. no. S2110; Beijing Solarbio Science & Technology Co.).

The primary antibodies used were: Anti-C5b-9 rabbit polyclonal antibody (pAb) (1:1,000; cat. no. ab55811; Abcam), anti-podocin rabbit monoclonal antibody (mAb) (1:500; cat. no. ab181143; Abcam), anti-beclin-1 rabbit mAb (1:50; cat. no. ab217179; Abcam), anti-LC3A/B rabbit antibody (1:200; cat. no. 4108; Cell Signaling Technology, Inc.), anti-SQSTM1 mouse mAb (1:100; cat. no. ab109012; Abcam), anti-β-catenin rabbit mAb (1:100; cat. no. 8480; Cell Signaling Technology, Inc.), anti-GSK-3β rabbit mAb (1:50; cat. no. 12456; Cell Signaling Technology, Inc.) and anti-Akt rabbit mAb (1:400; cat. no. 4691; Cell Signaling Technology, Inc. ). The secondary antibodies used were goat anti-rabbit IgG (H+L) Alexa Fluor 594 (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573","term_text":"R37117"}}R37117; Invitrogen; Thermo Fisher Scientific, Inc.) and goat anti-mouse IgG (H+L) Alexa Fluor 594 (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"R37121","term_id":"794577","term_text":"R37121"}}R37121; Invitrogen; Thermo Fisher Scientific, Inc.).