ILK affects protein kinase C α (PKC-α)–mediated phosphorylation of kindlin-3. (A) HL-60 cells were transfected with N-terminally green fluorescent protein (GFP)–tagged kindlin-3 and stimulated (stim) with interleukin-8 (IL-8) for indicated durations (100 nM), and GFP-trap–based immunoprecipitation was performed. Subsequent blotting with a serine phosphorylation–specific antibody revealed reduced phosphorylation of GFP-tagged kindlin-3 in ILK-deficient cells. Stimulation times (no stimulation [unstim ctrl], 30 seconds, and 3 minutes) are depicted above the representative image (n = 3-4). Analyses of chemokine (CXCL1)–induced arrest in mice treated with a PKC-α inhibitor or vehicle control (B) and of chimeric mice with PKC-α–deficient bone marrow (C) revealed reduced chemokine (CXCL1)–induced integrin-mediated leukocyte arrest in vivo (n = 3-5). For this purpose, mice were anesthetized, a carotid artery catheter was placed, and the mouse cremaster muscle was prepared. Upon direct observation via intravital microscopy, the chemokine CXCL1 was injected, and the arrest of leukocytes was assessed. (D) Analysis of isolated human neutrophils, stimulated with IL-8, showed a chemokine-dependent colocalization (colocalization channel in gray; left) of PKC-α (blue) and kindlin-3 (red) (n = 4). Images were recorded on a Zeiss LSM 700 confocal microscope equipped with a 63× oil immersion objective. (E) IL-8 chemokine stimulation of HL-60 cells led to membrane localization of PKC in control cells, whereas this translocation was absent in ILK-deficient cells (n = 3-5). (F) Furthermore, PKC activity as assessed by radioactivity measurement of substrate turnover revealed decreased PKC activity in ILK-deficient cells in control conditions vs stimulation for 3 minutes with IL-8 (n = 6). (G) Bone marrow from kindlin-3–deficient animals was transfected with different kindlin-3 mutants and subsequently differentiated as previously reported.²³  Cells were stimulated with CXCL1 and used for ICAM-1 binding assay. Lines above graph indicate significance among groups (n = 2-3 independent transfections; n ≥ 3 experiments). *P ≤ .05, **P < .01. FI, fold increase; IP, immunoprecipitation; KD, knockdown; KO, knockout; MFI, mean fluorescence intensity; n.s., not significant; SCR, scrambled.