2.11. Proteins isolation and Western blot

Protein in kidney tissues was extracted using RIPA (Solarbio, Beijing, China) and protein in cells was extracted using EpiQuik Whole Cell Extraction Kit (AmyJet Scientific, Wuhan, Hubei, China). Western blot analysis was performed following standard procedures. Protein lysates were separated in 10% sodium dodecyl sulphate‐polyacrylamide gels and electroblotted onto to a polyvinylidene fluoride membrane (Roche Diagnostics, Mannheim, Germany). Next, immunoblotting was carried out using antibody to Pdx‐1 (rabbit anti‐ Pdx‐1 antibody, 1:250; Abcam), Pax‐6 (rabbit anti‐ Pax‐6 antibody, 1:250; Abcam), insulin‐1 (rabbit anti‐ insulin‐1 antibody, 1:250; Abcam), Ngn3 (rabbit anti‐Ngn3 antibody, 1:250; Abcam), GK (rabbit anti‐GK antibody, 1:250; Abcam), nephrin (rabbit anti‐nephrin antibody, 1:400; Abcam), podocin (rabbit anti‐podocin antibody, 1:250; Abcam), CD2AP (rabbit anti‐CD2AP antibody, 1:250; Abcam), caspase‐3 (rabbit anti‐caspase‐3 antibody, 1:400; Beyotime, Shanghai), Bax (rabbit anti‐Bax antibody, 1:400; Beyotime, Shanghai), Bcl‐2 (rabbit anti‐Bcl‐2 antibody, 1:400; Beyotime, Shanghai), Notch1 (rabbit anti‐Notch1 antibody, 1:400; Beyotime, Shanghai), NICD (rabbit anti‐NICD antibody, 1:400; Beyotime, Shanghai), Hes‐1 (rabbit anti‐Hes‐1 antibody, 1:400; Beyotime, Shanghai), Delta (rabbit anti‐Delta antibody, 1:400; Beyotime, Shanghai), and β‐actin (rabbit anti‐β‐actin antibody, 1:800; Beyotime). Protein loading was determined using mouse anti‐β‐actin monoclonal antibody. Lab Works Image Acquisition and Analysis Software (UVP, Upland, CA, USA) were used to quantify band intensities. The analysis was carried out independently three times.