Fig 2: MLCK and myosin in myeloid cells.a Immunoblotting of primary murine myeloid cells (CD11b+) and Lewis lung carcinoma (LLC) cells with anti-myosin heavy chain A, B, and C or HSP90 antibodies. b RT-PCR analysis of mRNA expression of Myh9, Myh10, and Myh14 in myeloid cells (n = 3, *p < 0.0001 Myh9 vs Myh10 or Myh14). c Effect of Myh9 and Myh10 siRNA on Myh9 expression (n = 3, p = 0.0568). d, e Quantification (d) and FACS profiles (e) of VCAM-Fc binding to All Stars, Myh9 and Myh10 siRNA-transfected myeloid cells (n = 3). *p < 0.0001 for SDF-1 stimulated, p > 0.9999 for EBM stimulated, and p > 0.9000 for Mn2+ stimulated Myh9 vs All Stars and vs Myh10 siRNA-transfected cells. f Cell adhesion to VCAM of Myh9 and Myh10 siRNA-transfected myeloid cells (n = 3). *p < 0.0001 for SDF-1 and IL-1 stimulated Myh9 vs All Stars and Myh10 siRNA. g Effect of Myh9 and Myh10 siRNA on Rap1 activation by SDF-1a in myeloid cells. h Cytokine and p110?CAAX stimulation of myeloid cell adhesion to VCAM-1 in the presence of vehicle or the myosin ATPase inhibitor blebbistatin (n = 3). p = 0.9812 vehicle vs blebbistatin basal medium, *p < 0.0001 vehicle vs blebbistatin for SDF-1a, IL1ß, VEGF, p110?CAAX, and p = 0.9915 vehicle vs blebbistatin for pcDNA. i Immunofluorescence detection of integrin a4 (green) and myosin or Rap1 (red) in SDF1-bead-stimulated Mlck210-/- myeloid cells that were transfected with WT or kinase-dead Mlck210 cDNA. Nuclei were detected with Dapi (blue). Scale bar = 5 µm. All replicates are biologically independent samples. Data are presented as mean values ± SEM. Significance determined by one-way Anova with Tukey’s multiple comparisons for panels b, d, f, and by unpaired t-test for panels c, h. Experiments were performed twice. Source data are provided with this article as a Source Data file.

Fig 3: 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 PI3K? (p110?CAAX), or active Rap (RapG12V). p = 0.9791 untransfected WT vs Mlck210-/-, *p < 0.0001 G12VRas and p110?CAAX 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 IL1ß 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.