Rabbit anti wt1

Immunofluorescence was performed as previously described with minor modifications (Meng et al., 2019a (link)). Briefly, the cells were fixed with 4% Paraformaldehyde Fix Solution (Beyotime) for 20 min, and then permeabilized in Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime) for 10 min. After blocking with a blocking solution (Beyotime) for 20 min, the cells were incubated in the primary antibody at 4°C overnight. The primary antibodies used were rabbit anti-WT1 (1:200, Cell Signaling Technology, Inc.). The cells were washed with PBS and incubated with the Abclonal secondary antibody was incubated at 1:500 for 1 h. After washing, DAPI staining solution (Beyotime) was incubated for 3 min. After washing, the cells were observed using a fluorescence microscope.

Western blotting was performed as previously described with minor modifications (Meng et al., 2019a (link)). Briefly, the cells were washed twice with pre-cooled PBS, and RIPA buffer containing protease inhibitors was added to cells, which were lysed on ice for 20 min. The protein lysate was centrifuged at 4°C at 10,000 rpm for 5 min, and the supernatant was added to 5×SDS loading buffer at 100°C for 10 min. The denatured proteins were subjected to SDS-PAGE electrophoresis and were transferred to PVDF membranes (Millipore). After blocking with a blocking solution (Beyotime) for 20 min, the membrane was incubated in the primary antibody at 4°C overnight. The primary antibodies used were rabbit anti-WT1 (1:1,000, Cell Signaling Technology, Inc.), mouse anti-GAPDH, and mouse anti-ACTB (1:1,000, Abclonal). After washing, the Abclonal secondary antibody was incubated at 1:1,000 for 1 h. Finally, the ECL Luminescent Solution (Millipore) was used to visualize blots and for imaging of protein bands.