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.
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.