Fig. 4: PI3Kgamma activates MLCK210 and Rap-mediated adhesion.

a Akt phosphorylation in WT and Mlck210^−/− cytokine-stimulated murine myeloid cells. b Rap1 GTP loading in cytokine-stimulated WT and Mlck210^−/−murine myeloid cells. c Adhesion to VCAM of WT and Mlck210^−/− murine myeloid cells (n = 3) after transfection with active Ras (G12VRas), active PI3Kγ (p110γCAAX), or active Rap (RapG12V). p = 0.9791 untransfected WT vs Mlck210^−/−, *p < 0.0001 G12VRas and p110γCAAX WT vs Mlck210^−/−, p = 0.9245 RapV12 WT vs Mlck210^−/−. Significance testing by unpaired t-test. d Myosin Light Chain (MLC) S18/Thr19 phosphorylation in basal or cytokine-stimulated WT and Mlck210^−/− murine myeloid cells. e Immunoblotting to detect Flag-tagged MLCK210, total MLCK210 and HSP90 in WT and Mlck210^−/− murine myeloid cells transfected with control (pcDNA) and MLCK210 expression plasmids. f Immunoblotting to detect phosphorylated MLC and total MLC in SDF1a stimulated or unstimulated WT or Mlck210^−/− myeloid cells that had been transfected with control (pcDNA) or flag-tagged WT and Kinase-dead (KD) MLCK210 expression plasmids. Graph: Relative pS18/Thr19-MLC expression in 3 independent samples. g Cytokine-stimulated VCAM-1 adhesion of WT and Mlck210^−/− myeloid cells transfected with control (pcDNA), WT or Kinase Dead (KD) MLCK210 expression constructs (n = 3). *p = 0.0001 for cytokine-stimulated Mlck210^−/− cells and Mlck210−/− cells expressing Mlck210KD cDNA vs WT cells. No significant differences (ns) for WT vs Mlck210^−/− cells expressing Mlck210 WT cDNA, p = 0.9936 for IL1β and p > 0.9999 for SDF-1a and VEGF-A treated cells. No significant differences (ns) for basal conditions (p > 0.9999). Significance determined by unpaired t-test for 4c and by one-way Anova with Tukey’s multiple comparisons for panels e, g. All replicates are biologically independent samples. Experiments were performed twice. Data are presented as mean values ± SEM. Source data are provided with this article as a Source Data file.