Clathrin interacts with clathrin box sequence within the F3 domain of kindlin-2. (A) Representative binding of 1 concentration (1.6 μM) of the following full-length kindlin-2 (1-680) and kindlin-2 fragments: (1-105), (95-680), and (281-541) to clathrin TD domain (1-363). The clathrin TD domain was immobilized on the CM5 sensor chip surfaces (∼500 RU). (B) EGFP-tagged WT and mutants of kindlin-2 were immunoprecipitated from lysates of CHO cells transfected with the respective kindlin-2 constructs (as shown) using GFP-Trap Sepharose. The binding of clathrin to kindlin-2 mutants was analyzed on western blots with Abs to CHC. Kindlin-2 expression levels are revealed with anti-GFP Ab. The results are representative of 3 independent experiments. (C) Kindlin-2 was immunoprecipitated from WT ECs in the absence or presence of a peptide corresponding to residues 584-604 of kindlin-2 or its scrambled version followed by western blot analysis with anti-CHC and anti–kindlin-2 Abs. The images are representative of 2 experiments. (D-E) Overexpression of ΔF3 or Δ(LIRMD) deletion mutants of kindlin-2 does not reduce surface expression of CD39 and CD73 in kindlin-2^+/− ECs. The cells were transfected with the EGFP constructs of WT, QW/AA⁶¹⁵, deletion mutants of kindlin-2, or empty EGFP vector as indicated. Cell-surface expression of CD39 (D) and CD73 (E) were measured using PE-conjugated anti-CD39 and anti-CD73 Abs by FACS analysis of the EGFP⁺ cell population. The data represent mean ± SEM of triplicate samples and are representative of 3 independent experiments.