Anti src py418

Cells were washed with PBS and lysed with n-octyl-β-D-glucoside (ODG) buffer [20 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 1% Nonidet P-40, 5% glycerol, 2% ODG, and a protease inhibitor cocktail], and immunoblotting was carried out as described previously [26] (link). The following primary antibodies were used: anti-Src (Ab-1; Calbiochem), anti-Src pY418 (Invitrogen), anti-phosphotyrosine (4G10; Millipore), anti-cortactin (Sigma), anti-cortactin pY421 (Sigma), anti-FAK (Santa Cruz), anti-FAK pY576 (Cell Signaling), anti-ERK pT202/Y204 (Cell Signaling), anti-ERK (Cell Signaling), anti-GAPDH (Sigma), anti-Flottilin-1 (BD) and anti-Caveolin-1 (BD). Anti-Cbp and anti-Cbp pY314 were prepared as described previously [19] (link). GAPDH was used as a loading control. Horseradish peroxidase (HRP)–conjugated anti-mouse or anti-rabbit IgG (Zymed) was used as the secondary antibody. All blots were visualized and quantitated using a LAS-4000 luminescent image analyzer (GE Healthcare).

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).