Rabbit anti pcna

Total protein was extracted by treating collected cells with RIPA lysate (Solarbio, Beijing, China) containing protease inhibitors, followed by measuring the protein concentration using a BCA protein concentration assay kit (Solarbio, Beijing, China). Equal protein samples were resolved using 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Next, membranes were blocked with 5% skimmed milk for 2 h and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: FOXO1 (Rabbit anti-FOXO1, ABclonal, Wuhan, China), PCNA (Rabbit anti-PCNA, Proteintech, Wuhan, China), CCND1 (Rabbit anti-CCND1, Proteintech, Wuhan, China), CDK2 (Rabbit anti-CDK2, Bioss, Beijing, China), MYOD (Rabbit anti-MYOD, Bioss, Beijing, China), MYOG (Rabbit anti-MYOG, ABclonal, Wuhan, China), MYHC (Rabbit anti-MYHC, ABclonal, Wuhan, China), and ACTB (Rabbit anti-ACTB, Proteintech, Beijing, China). On the next day, membranes were washed three times with Tris-buffered saline with Tween-20 (TBST) buffer and incubated with HRP-labelled secondary antibodies for 2 h at 37 °C. Membranes were washed three times with TBST and visualized using ECL (NeoSami, Suzhou, China) ultrasensitive luminescence. Finally, images were captured using the ChemiDocXRS system, and the bands were analyzed in greyscale using Image J software.

Frozen tissue sections from control and PCOS rat model were fixed in 4% paraformaldehyde at room temperature following with 20% sucrose for 48 h. The tissues were embedded with OCT and sliced at −20 °C. After fixed with acetone, goat serum was used to block at room temperature for 2 h. The primary antibodies mouse anti-NPRA (1:50; Santa Cruz Biotechnology, CA, USA) and NPRC (1:100; Santa Cruz Biotechnology, CA, USA), rabbit anti-PGRMC1 (1:100; Cell Signaling, Danvers, MA, USA) and PGRMC2 (1:50; Cell Signaling) were incubated at 37 °C for 2 h. Washing with PBS, TRITC-conjugated goat anti-rabbit IgG (1:100; ZSGB-BIO, Shanghai, China) and FITC-conjugated goat anti-mouse IgG (1:100; Sigma-Aldrich, St. Louis, MO, USA) were incubated for 1 h at 37 °C following treatment with DAPI (1:1000; Beyotime, Shanghai, China) for 3 min. The immunofluorescent images were taken with an inverted microscope (Olympus, Tokyo, Japan).
Cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature following incubation with 0.1% Triton X-100 for 15 min. After blocking with goat serum, cells were incubated with rabbit anti-PCNA (1:200; Proteintech, Wuhan, China) at 4 °C overnight. TRITC-conjugated goat anti-rabbit IgG was incubated for 1 h and DAPI (1:1000) for 3 min. Immunofluorescent staining was photographed with an inverted microscope.

The renal tissue and cells are lysed on ice with lysis buffer, and protein concentration was determined. The samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PVDF membrane (Microporous, USA). Subsequently, the membrane was blocked for 2 h, and probed with primary antibody at 4°C, such as rabbit-anti-fibronectin (1:1,000), rabbit-anti-Shh (1:1,000; Proteintech), rabbit-anti-Shh (1:500; Santa Cruz), rabbit-anti-TNF-α (1:1,000; Santa Cruz), rabbit-anti-MCP-1 (1:1,000, Proteintech), rabbit-anti-PCNA (1:1,000; Proteintech), rabbit-anti-Vimentin (1:1,000; Proteintech), mouse-anti-p21 (1:1,000; Santa Cruz), mouse-anti-p16^INK4A (1:1,000; Santa Cruz), rabbit-anti-PDGFR-β (1:1,000; Santa Cruz), rabbit-anti-β-actin (1:8,000; Proteintech), and anti-GAPDH antibody (1:5,000; Wuhampmack Biotechnology Co., Ltd., China) for 16 h at 4°C. The chemiluminescence signal was detected after incubation with second antibody for 1 h. Finally, ImageJ software were used for analysis.