Anti fyn antibody

For co-immunoprecipitation assay, collected cells were lysed in the lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40, 1 mM sodium fluoride and 1 mM sodium orthovanadate. Insoluble proteins (500 μg per sample) were incubated with anti-Fyn antibody (Santa Cruz, sc-16) and Protein A/G Plus Agarose beads (Santa Cruz) for 14 h at 4°C with constant rotation. Immunocomplexes were washed four times with 0.1% Triton-100 in PBS and eluted by boiling in the Laemmli buffer. Primary antibodies used for immunoblot were listed as follows: anti-PTPRZ1 (BD Biosciences, 610179, 1: 1,000); anti-SOX2 (Bethyl Laboratories, A301-739A, 1: 1,000); anti-Phospho-AKT (p-Ser473, Cell Signaling, #4060, 1: 2,000); anti-AKT (Cell Signaling, #2966, 1: 500); anti-CD133 (Millipore, MAB4399, 1: 500); anti-GFAP (Covance, PRB-571C, 1: 1,000); anti-MAP2 (Covance, SMI-52R, 1: 1,000); anti-p-SFK (Tyr416, Cell Signaling, #6943, 1: 500); anti-Fyn (Cell Signaling, #9320, 1: 500); anti-Tubulin (Sigma, T9026, 1: 10,000); and anti-GAPDH (Cell Signaling, #2118, 1: 2,000). Representative uncropped immunoblots were presented in Supplementary Fig. 12. All immunoblot results were independently repeated for at least three times.

Podocytes were lysed in radioimmunoprecipitation assay (RIPA) buffer at 4°C for 10 minutes. Equal amounts of podocyte lysate were incubated with anti-Fyn antibody (Santa Cruz Biotechnology, CA, USA) at room temperature for 2 hours. Immunocomplexes were isolated using Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, CA, USA) and recovered by boiling for 3 minutes in 1x electrophoresis buffer. Proteins were eluted with SDS-PAGE sample buffer and immunoblotted with anti-Src [PY418] (Invitrogen, CA, USA).