Iqgap1 rabbit pab

The protein concentration was measured using a BCA protein assay (Thermo Scientific, USA). Equal amounts of boiled protein in loading buffer were separated using NuPAGE 10% Bis-Tris SDS-PAGE (Life Technologies, USA) and then electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with primary antibodies (nephrin rabbit pAb, 1:200, Abcam, UK; p-nephrin rabbit monoclonal antibody [mAb], 1:10,000, Abcam, UK; IQGAP1 rabbit pAb, 1:200, Santa Cruz, USA; p-serine [p-Ser] mouse mAb, 1:300, Millipore, USA; Myc mouse mAb, 1:2000, California Bioscience, USA; β-actin mouse mAb, 1:1000, Santa Cruz, USA) overnight at 4 °C, followed by incubation with a secondary antibody (LICOR, Lincoln, NE, USA), and the membranes were then analyzed using the ODESSEY infrared imaging system (LICOR, Lincoln, NE, USA). The integrated optical density for each band was calculated using a Gel-Pro Analyzer, version 4.5.

Co-immunoprecipitation experiments were conducted according to the manufacturer’s instructions (P2012, Beyotime, China). The total protein of the cultured podocytes or COS7 cells was extracted using lysis buffer (1.0% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 20 mM Tris, pH 7.5) that contained a protease inhibitor cocktail (P8340, Sigma-Aldrich, USA). The samples were centrifuged at 13,000 g for 5 min at 4 °C. Nephrin rabbit pAb (2 μg/500 μg total protein; Santa Cruz, USA) or IQGAP1 rabbit pAb (2 μg/500 μg total protein, Santa Cruz, USA) was added to the supernatant and rotated overnight at 4 °C. Then, the mixture was loaded with protein A + G agarose and incubated for 3 h at 4 °C. The centrifuged sediment was saved and mixed with 1× LDS sample buffer. After boiling at 70 °C for 10 min, the samples were analyzed by Western blotting.