Defective clathrin-dependent trafficking of CD39 and CD73 in kindlin-2^+/−ECs. (A) Western blot analysis of WT and kindlin-2^+/− EC lysates probed with Abs to CD39 and CD73 reveal similar total content of these enzymes in both mouse strains (top panel). The blots were reprobed with Ab to actin to confirm equal protein loading (bottom panel). (B-C) Comparison of kinetics of CD39 (B) and CD73 (C) internalization in WT and kindlin-2^+/− ECs. The measurements were performed as described in “Methods.” The data are mean ± SEM of triplicate samples and are representative of 3 independent experiments. (D) Kindlin-2 and clathrin colocalization in WT ECs. The cells were stained with mAbs to kindlin-2 followed by Alexa 568–coupled goat anti-mouse IgG (red fluorescence) and rabbit anti-clathrin Ab and Alexa 488–conjugated goat anti-rabbit IgG (green fluorescence). Immuno-Fluore mounting medium (MPI Biomedicals) was used to mound the slides, and the images were taken with a 63 × 1.4 oil objective using a Leica TCS-NT laser scanning confocal microscope and Leica confocal software (version 2.5, build 1227). Images are representative of 3 independent experiments (bar size, 20 μm). Arrows point to focal adhesions. (E) Clathrin coimmunoprecipitates with kindlin-2 from EC lysates. Kindlin-2 was immunoprecipitated from lysates of MAECs (left panel) and HUVECs (right panel). Immunoprecipitates were analyzed on western blots with Abs to CHC and kindlin-2 as indicated. Images are representative of 3 independent experiments. (F) The CTD (1-363) was immobilized on the CM5 sensor chip surfaces (500 RU). Sensograms obtained for a concentration series of GST-kindlin-2 or GST protein (each concentration measured twice and shown as colored lines) were fit to a 1:1 interaction model with a drifting baseline (shown as black lines). Ig, immunoglobulin; IP, immunoprecipitation; RU, response units.