Ab152126

The total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with phosphatase and protease inhibitors (KeyGEN BioTECH; Nanjing, China) and quantified using Enhanced BCA Protein Assay Kit (Beyotime). Subsequently, 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to separate equal amounts of proteins, followed by their transfer onto a polyvinylidene fluoride (PVDF) membrane. Nonspecific proteins were blocked with 5% skim milk at room temperature for 2 h. After overnight incubation with a primary antibody, anti-E2F3 (1:1,000 dilution; ab152126) or anti-GAPDH (1:1,000 dilution; ab128915; Abcam, Cambridge, UK), the PVDF membrane was incubated with a secondary antibody (Abcam) for 2 h. Protein blots were subsequently visualized using BeyoECL Plus Kit (Beyotime).

Protein expression levels of E2F3, Bcl-2 related X protein (Bax), B-cell
lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1),
C-caspase3, C-caspase7, and C-caspase9 were measured using western blot assay in
podocytes under different treatments. Transfected podocytes (2×10⁶cells/well) were lysed using pre-cold RIPA buffer (Beyotime Institute of
Biotechnology) according to the manufacturer instructions. Podocyte lysates were
separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then isolated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane (Millipore, USA). After blocking the proteins with 5%
non-fat milk in the membranes for 1 h at room temperature, membranes were
incubated with primary antibodies against E2F3 (ab152126, 1:2000 dilution), Bad
(ab32445, 1:1000 dilution), Bcl-2 (ab32124, 1:1000 dilution), Bax (ab69643,
1:1000 dilution), APAF1 (ab2001, 1:5000 dilution), C-caspase3 (ab4051, 1:1000
dilution), C-caspase7 (ab32522, 1:1000 dilution), C-caspase9 (ab2014, 1:1000
dilution), and GAPDH (1:1000, ab9485) (all from Abcam, UK) overnight at 4°C. The
horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1:10,000
dilution, Abcam) was then incubated with the membranes at room temperature for 1
h. Finally, an ECL detection kit (Pierce Biotechnolgy, USA) was used to detect
the protein bands.

Total proteins were extracted from cells using lysis buffer, and the concentrations of proteins were measured by the BCA method. 12% SDS-PAGE was utilized to separate proteins and then transferred onto PVDF membranes. Then, 5% skimmed milk was used to block membranes for 50 min at room temperature followed by incubation with primary antibodies at 4°C overnight. The primary antibodies are listed as follows: anti-cyclin D1 (1 : 1000; ab16663; Abcam, Shanghai, China), anti-p21 (1 : 1000; ab109520; Abcam, Shanghai, China), anti-Bax (1 : 1000; ab32503; Abcam, Shanghai, China), anti-Bcl-2 (1 : 1000; ab32124; Abcam, Shanghai, China), anti-cleaved-caspase-3 (1 : 1000; ab2302; Abcam, Shanghai, China), anti-cleaved-caspase-9 (1 : 1000; ab2324; Abcam, Shanghai, China), anti-Cox-2 (1 : 1000; ab179800; Abcam, Shanghai, China), anti-MMP-2 (1 : 1000; ab92536; Abcam, Shanghai, China), anti-MMP-9 (1 : 1000; ab76003; Abcam, Shanghai, China), anti-E2F3 (1 : 1000; ab152126; Abcam, Shanghai, China), and anti-GAPDH (1 : 1000; ab8245; Abcam, Shanghai, China). The membranes were then incubated with secondary antibody (1 : 1000; ab7090; Abcam, Shanghai, China) on the next day at 37°C for 2 h. An enhanced chemiluminescence kit and the ImageJ software were applied to protein quantification.

Total proteins were isolated from cells using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and quantified using a BCA protein detection kit (Beyotime, Shanghai, China). Then, 30 μg of protein sample from each group was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the PVDF membranes were blocked with 5% skimmed milk and then incubated with anti-E2F3 antibody (1:1000, ab152126, Abcam Inc., Cambridge, UK) and anti-GAPDH antibody (1:1000, ab9485, Abcam Inc., Cambridge, UK) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:2000, ab150077, Abcam Inc., Cambridge, UK) for 1h at room temperature. Next, the protein bands were developed using an ECL chemiluminescence substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were washed with PBS and subjected to a lysis buffer. Protein lysates were separated using 15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with a buffer containing 10% non-fat milk in PBS with 0.05% Tween-20 for 2 h and incubated with E2F3 antibody (1:1000, ab152126, Abcam), anti MMP-2 antibody (1:1000, ab97779, Abcam) and β-actin (1:1000, PAB36265, Bioswamp). Then the membranes were incubated with specific secondary antibodies attached to horseradish peroxidase at 4°C. Evaluation of the expression of proteins was performed using Image J version 1.38.