Ab180579

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).

Rabbit anti‐Aurora B antibodies (ab2254), rabbit anti‐MAD2L2 [EPR13657] antibodies (ab180579), rabbit anti‐phospho‐histone H3 (Ser10) antibodies and rabbit anti‐phospho‐histone H2AX (phospho‐S139) antibodies (ab2893) were purchased from Abcam (Cambridge, UK). AZD1152‐HQPA was purchased from Abcam. Mouse anti‐Aurora B antibodies (3F11) were obtained from R&D (Chicago, IL, USA). Anti‐Aurora B (phospho‐Thr232) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti‐rabbit Alexa Fluor 488 and goat anti‐mouse IgG H&L (Alexa Fluor 647) were obtained from Abcam. Human tubal fluid (HTF) was obtained from Sage Science (Beverly, MA, USA). 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) (10 μg/mL) was obtained from Solarbio (China).

Kidney tissues were transferred to 4% paraformaldehyde and fixed at 4 °C overnight. 4-μm paraffin embedded sections were deparaffinized and rehydrated for immunohistochemical staining. The sections were then incubated with the primary antibody for MAD2B (1:100, ab180579, Abcam, Cambridge, MA, USA), Numb (1:200, #2756, Cell Signaling Technology, Danvers, MA, USA) and WT1 (1:100, ab88901, Abcam, Cambridge, MA, USA) overnight at 4 °C. After being stained with hematoxylin, the sections were observed under a light microscope. We calculated the positive stained areas per glomerulus using Image-Pro Plus 6.0 software.

Cells were lysed in 2% SDS, 1% NP-40, 50 mM Tris pH 7.5, and 150 mM NaCl. Protein-lysates were equalized based on protein concentration measured using the BCA Protein Assay Reagent (Thermo Fisher Scientific). Protein samples were separated on Novex 4–12% Bis-Tris gradient gels using MOPS SDS running buffer and NuPage LDS sample buffer (all Thermo Fisher Scientific); subsequently, proteins were transferred onto Immobilon‐FL membranes (Merck Millipore). The following primary antibodies were used: Anti-PARP1 (rabbit polyclonal, Cell Signalling Technology #9542, 1:1000), anti-OBFC1 (STN1, mouse monoclonal, Santa Cruz sc-376450, 1:1000), anti-53BP1 (rabbit polyclonal, NOVUS biologicals NB100-304, 1:1000), anti-Mad2L2/REV7 (rabbit monoclonal, Abcam ab180579, 1:1000), anti-Cas9 (mouse monoclonal, 7A9‐3A3 Cell Signaling Technology, 1:1000) and anti-Tubulin (mouse monoclonal, Sigma-Aldrich, T6199 clone DM1A, 1:5000). The secondary antibodies CF680 goat anti‐rabbit IgG and CF770 goat anti‐mouse IgG (Biotium, both at 1:10.000) and the Odyssey CLx infrared imaging scanning system (LI‐COR Biosciences) were used to detect protein expression.