Rab18 influences FA maturation and FAK phosphorylation. (a) U2OS cells cotransfected with Vinculin-GFP and DsRed-ER imaged before (prebleach), during bleaching, and after photobleaching of one adhesion positive for Vinculin-GFP (white circle). Scale bar: 20 µm; inset: 4 µm. (b and c) Total linear fitted recovery curves with standard derivation for Vinculin-GFP or β3-integrin-YFP in control cells, cells silenced for Rab18, or cells silenced for Rab18 and transfected with RFP-Rab18. Curves show the recovery for adhesion points in contact with the ER (ER⁺; b) or not (ER⁻; c). For each curve, the value of the MF ± SEM is reported as percentage. A total of 27 adhesion points were bleached for each condition, in three independent experiments. (d) U2OS cells transfected with siRNA control or siRNA against Rab18, or depleted for Rab18 and subsequently transfected with GFP-Rab18, were fixed and stained with anti-pY397-FAK and anti-vinculin antibodies. Insets in the top right show magnifications of the boxed areas for pY397-FAK. Insets in the bottom right show the same adhesion with a color-coded map of the pY397-FAK mean fluorescence intensity. GFP channel for the cell silenced for Rab18 and transfected with GFP-Rab18 is shown in the upper right corner inset. Scale bar: 5 µm; inset: 1 µm. (e) Quantification of FAK activation on FAs measured as mean fluorescence intensity of pY397-FAK per adhesion. The values represent the mean ± SEM of all the adhesions in a cell from three independent experiments (n > 30 cells). *, P < 0.05; **, P < 0.01. (f) Lysates from U2OS cells transfected with siRNA control, siRNA Rab18, or silenced for Rab18 and subsequently transfected with GFP-Rab18 were subjected to Western blot analysis with antibodies against FAK, pY397-FAK, Rab18, and tubulin (as loading control). (g) Quantification of FAK phosphorylation normalized to the levels of FAK and plotted relative to the control cells is shown as mean ± SEM from three independent experiments. *, P < 0.05.