TSR induces SHP-1 association with CD36 and VEGFR2 in MVEC. (A) MVEC were cultured to confluence and then treated with TSP-1, recombinant TSR, or thioredoxin (Thio) control for 4 hours before exposure to 50 ng/mL VEGF for 5 minutes. VEGFR2 phosphorylation was analyzed by western blot with an antibody to VEGFR2 pTyr1175. Blots were reprobed with anti-VEGFR2 as a loading control. VEGFR2 phosphorylation levels are expressed as the ratio of phosphorylated VEGFR2 to total VEGFR2. (B-C) MVEC were treated with TSR (10 nmol/L) in low-serum medium for 4 hours before exposure to VEGF (50 ng/mL) for 15 minutes. Cell lysates were then immunoprecipitated with anti-VEGFR2 (B) or anti-CD36 (C), and coprecipitated proteins were detected by western blot analysis with antibodies to SHP-1, SHP-2, phospho-SHP-2, phospho-VEGFR2, VEGFR2, or CD36. Blots are representative of 3 experiments. (D) Immunofluorescence images of MVEC treated with 10 nmol/L TSR and/or 50 ng/mL VEGF and then probed with mouse monoclonal anti–SHP-1 and rabbit polyclonal anti-CD36, followed by Alexa Fluor 488–conjugated anti-mouse IgG and Alexa Fluor 594–conjugated anti-rabbit IgG. The top row shows Alexa Fluor 488 images, the middle row shows Alexa Fluor 594 images, and the bottom row shows the merged images.