Ab4147

The total protein of cultured cells and isolated renal glomeruli were extracted with RIPA lysis buffer (Beyotime, Shanghai, China). The total protein lysates were measured by a BCA protein assay kit (Beyotime). After being boiled for 5 min at 95 °C in SDS protein loading buffer, proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked in 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. The following primary antibodies against the following targets were used in this study: MAD2B (1:1000, ab180579, Abcam), Numb (1:1500, ab4147, Abcam), NICD (1:1000, ab52627, Abcam), Hes-1 (1:1000, ab71559, Abcam), Desmin (1:1000, BS1712, Bioworld Technology, Louis Park, MN, USA), MDM2 (1:1000, ab16895, Abcam), active β-catenin (1:1000, #4270, Cell Signaling Technology, Danvers, MA, USA), β-actin (1:10000, sc-47778, Santa Cruz, CA, USA) and α-tubulin (1:3000, Protein Tech Group, Chicago, IL, USA). Finally, the membranes were incubated with horseradish secondary antibodies and detected by an ECL system (Thermo). The densitometric analysis of western blot images was measured by ImageJ software (National Institutes of Health).

Protein extracts for Western blot analysis were obtained as described previously [17 (link)]. For Western blot detection, we used NUMB (ab4147, Abcam, 1 μg/mL) and NUMBL (ab37500, Abcam, 1 μg/mL) antibodies. α-Tubulin (T9026, Sigma) was used as a control. Horseradish peroxidase-labeled rabbit anti-mouse (ab97046, Abcam, diluted 1:5,000) and goat anti-rabbit (ab97051, Abcam, diluted 1:5,000) secondary antibodies were used.