eIF6 regulates the spatial mechanoactivation of ERK1/2. ECs transfected with si Scr or si eIF6 were exposed to force for 0, 5, or 30 min. (a and b) Representative Western blots of pERK^T202/Y204 and t-ERK1/2 and quantification (n = 4). (c–e) ECs were transfected with si Scr or si ERK1/2 (d), and following siRNA-mediated knockdown, ECs were transiently transfected with plasmids encoding WT ERK1/2 (p.ERK1/2) or CA-ERK (p.CA-ERK; e). (c, f, and g) Mechanical force was applied for 0 min (no force [NF]) or 30 min (force [F]) to p.ERK1/2- and p.CA-ERK–expressing ECs. Representative superresolution immunofluorescent micrographs showing pERK^T202/Y204 (green) and focal adhesions (vinculin; red) in ECs following force (c); PECAM-1–coated beads are highlighted by white circles. Scale bars = 20 μm. Quantification of mean frequency per cell (f) and mean area (g) of vinculin-positive focal adhesions following force for 0 min (NF) or 30 min (F; n > 30 cells across three separate experiments). (h) Representative superresolution immunofluorescent micrographs showing colocalization of pERK^T202/Y204 (green) with focal adhesions (vinculin, red) following application of force. Larger images are higher magnification images of indicated region of whole cells shown in smaller images. Magnetic beads are highlighted by white circles. Scale bars = 20 μm. (i) Quantification of mean fluorescence intensities of pERK^T202/Y204 following application of force (n > 30 cells across three separate experiments). (j) Image analysis quantification of colocalization of pERK^T202/Y204 with focal adhesions, using Pearson’s coefficient, following force (n > 30 cells across three separate experiments). Values in b, f, g, i, and j are mean ± SEM, and significance was determined by two-way ANOVA. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. FA, focal adhesion. Source data are available for this figure: SourceData F5.