Paxillin and closely related LIM-domain proteins localize to clustered integrin-β1 or -β3 ct.

<p>(<b>A</b>) Schematic overview of the OPTIC workflow. Opa-expressing Ngo are used to cluster CEACAM3-integrin β cytoplasmic tail fusion proteins potentially resulting in the recruitment of an intracellular protein of interest (POI). (<b>B</b>) 293T cells were transiently cotransfected with a CEACAM3-ITGB1 (ITGB1) or CEACAM3-ITGB3 (ITGB3) fusion construct together with GFP-labelled LIM-domain-containing proteins. WCLs of the transfected 293T cells were probed by western blotting with a monoclonal antibody against GFP to detect the expression of GFP-LIM proteins (upper panel). Coomassie staining (lower panel) was used to verify equal loading of the membrane. (<b>C</b>) 293T cells transfected with CEACAM3-ITGB1 (ITGB1) and the indicated GFP-fusion proteins were seeded on poly-L-lysine. Cells were infected for 1 h with Pacific Blue−labelled <i>Neisseria gonorrhoeae</i> (Ngo; blue), fixed, and stained for ITGB1 (red). Recruitment of GFP-LIM proteins to clustered ITGB1 tail is indicated by white arrowheads. Bars represent 2 μm. (<b>D</b>) Quantification of (C). Each data point reflects the recruitment ratio R in a CEACAM3-ITGB1-expressing cell with associated bacteria. Horizontal lines indicate mean values and 95% confidence intervals (whiskers) of <i>n</i> = 60 cells from 3 independent experiments. Statistical significance was calculated using one-way ANOVA, followed by Bonferroni multiple comparison test (*** <i>p</i> < 0.001, ns = not significant). The data underlying this panel can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002757#pbio.3002757.s006" target="_blank">S1 Data</a>. (<b>E</b>) Quantification and statistical evaluation as in (D) of GFP-fusion protein recruitment to CEACAM3-ITGB3 (ITGB3) (see main <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002757#pbio.3002757.g001" target="_blank">Fig 1A</a>). The data underlying this panel can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002757#pbio.3002757.s006" target="_blank">S1 Data</a>.</p> <p>(PDF)</p>