Identification and characterization of leupaxin as new kindlin-3 interactor. (A) Domain structure of leupaxin. A C-terminal leupaxin fragment bound to kindlin-3 in a yeast-two-hybrid screen. (B) Flag-immunoprecipitation (IP) from lysates of +/+ and Flag-tagged kindlin-3 expressing (Flag/Flag) bone marrow–derived macrophages to verify interaction with endogenous leupaxin. (C) Western blot analyses of leupaxin, talin, and paxillin expression in +/+ RAW cells and four different K3^−/− RAW cell clones. (D) Domain structure of kindlin-3. (E) GFP-IP from lysates of K3^−/− RAW cells expressing EGFP, EGFP-K3, or the EGFP-tagged K3 QA mutant analyzed for leupaxin. (F) GFP-IP from lysates of K3^−/− RAW cells expressing different EGFP-K3 fragments to identify the leupaxin-interacting domain. (G) GFP-IP from lysates of K3^−/− RAW cells expressing EGFP, EGFP-K3, EGFP-K3 M3 mutant, or EGFP-K3 F1–3 to investigate the interaction with leupaxin and paxillin. (H) Western blot analyses of +/+ RAW cells and two leupaxin^−/− RAW cell clones for their expression of kindlin-3, paxillin, PTP-PEST, and talin. (I) GFP-IP from lysates of leupaxin^−/− RAW cells reconstituted with EGFP-tagged FL leupaxin (LPXN), a N-terminal fragment (NT), or its CT to determine interaction with kindlin-3. (J) GFP-IP from lysates of leupaxin^−/− RAW cells reconstituted either with EGFP-tagged WT leupaxin or a leupaxin mutant (C293R) analyzed for kindlin-3 binding. (K) GST-pulldown with GST, GST-leupaxin FL, GST-leupaxin NT, and GST-leupaxin CT incubated with His-Sumo-tagged K3 F0.