Fig. 1: MLCK210 is an integrin α4β1-associated protein.

a Integrin α4β1 immunoprecipitates from tumor conditioned medium (TCM) stimulated or unstimulated (Basal) CD11b+ myeloid cells were silver-stained or immunoblotted to detect co-precipitating proteins. (Left) Integrin α4, paxillin, talin, and IgG were detected in α4 integrin immunoprecipitates by immunoblotting. Right: Co-immunoprecipitated integrin α4, 210 kDa (MLCK210) and 80 kDa (Gelsolin) proteins as well as a 50 kDa protein (IgG) were detected in α4 integrin immunoprecipitates by silver staining. These samples derived from the same experiments and silver-stained gels and immunoblots were processed in parallel. b Spectra and peptide sequences from tandem mass spectroscopy analysis of 80 kDa and 210 kDa proteins co-immunoprecipitated with integrin α4β1; these proteins were identified as gelsolin and a high molecular weight isoform of myosin light chain kinase, MLCK210, respectively. c Plasma membranes from unstimulated and SDF-1α stimulated WT and Mlck210^−/− myeloid cells immunoblotted to detect MLCK210 and integrin α4. d Representative immunofluorescence images of integrin α4 (green) in BSA or SDF-1α stimulated WT and Mlck210^−/− myeloid cells. Scale bars = 10 µm. e Proportion of clustered integrin α4β1 in individual WT cells stimulated with BSA (n = 126 cells) or SDF-1α (n = 73 cells) and in individual Mlck210^−/− myeloid cells stimulated with BSA (n = 36 cells) or SDF-1α (n = 28 cells). p = 0.9247 WT BSA vs Mlck210^−/− BSA, p < 0.0001 WT SDF-1α vs Mlck210^−/− SDF-1α. Replicates are biologically independent samples. Statistical significance determined by one-sided Anova with Tukey’s multiple comparisons. f Detection of MLCK210 and loading control HSP90 in immunoblots of lysates from Mylk (Mlck) and All Stars control siRNA-transfected myeloid cells. g Images of integrin α4 (green, arrows) in SDF-1α stimulated Mylk (Mlck) and non-silencing siRNA-transfected myeloid cells. h Images of SDF-1α stimulated Mlck210^−/− myeloid cells transfected with wildtype (WT) or kinase dead (KD) Flag-tagged MLCK210 immunostained to detect integrin α4 (green) and MLCK210 (red). Nuclei were detected with Dapi (blue). Graph: Percent of cells with co-clustered integrin α4 and MLCK210 (n = 8 WT BSA; n = 13 WT SDF; n = 8 Mlck210^−/− BSA; n = 4 Mlck210^−/− SDF where n = experiments). p = 0.6999 WT BSA vs Mlck210^−/− BSA; p < 0.0001 WT SDF vs Mlck210^−/−SDF. Statistical significance determined by one-sided Anova with Tukey’s multiple comparisons. Scale bars = 5 µm. Data are presented as mean values ± SEM. Experiments were performed twice except for mass spectrometry, which was performed once. Source data are provided with this article as a Source Data file.