α tubulin antibody

Protein samples from kidney tissue or cell culture were lysed in radioimmunoprecipitation assay buffer with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysates were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% BSA/TBS-0.5% Tween-20 followed by incubation with primary antibodies overnight at 4°C. For immunoprecipitation, tissue lysates were incubated with anti-TIE2 antibody and subsequently with Dynabeads against Protein A (Invitrogen). The samples were eluted with Laemmli sample buffer with a reducing agent containing 200 mM dithiothreitol. The following antibodies were used for Western blot analysis: TIE2 antibody (Santa Cruz; C-20; SC-324), 4G-10 platinum anti-phosphotyrosine (EMD Millipore; 05–1050), α-tubulin antibody (Genscript; A01410), eNOS (Cell Signaling Technology; 49G3), phospho-eNOS (Cell Signaling Technology; C9C3), and FOXO1 (Cell Signaling Technology; C29H4). HRP-tagged secondary antibodies (Jackson ImmunoResearch) were used in a dilution of 1:10,000 in TBS-0.5% Tween. Quantification of protein expression was performed on multiple replicate experiments using the software ImageJ.