Vinculin knockdown prevents HGF-induced pMLC accumulation near cell–cell junctions. (A) HGF (2 h) induced increase in pMLC near cell–cell junctions revealed by IF. (B) A magnified view of A, showing that pMLC accumulates at F-actin structures that connect to p120-labeled cell–cell junctions. (C) IF shows a reduction of pMLC recruited to cell–cell junctions in vinculin knockdown (KD) cells after 2 h of HGF. (D) Overlay of the vinculin channel (smoothed with a Gaussian; r = 40) and pMLC channel of a dual-color IF staining of vinculin knockdown cells after 2 h of HGF. (E, left) Representative image automatically generated by custom software, displaying the line fragments used to measure pMLC levels near cell–cell junctions (green) and in the cytoplasm (red). (right) The relative intensity of pMLC near cell–cell junctions (a value of 1 means equal levels) shows an increase after 2 h of HGF that is largely abolished by vinculin knockdown. Error bars represent SEM. 25 images were analyzed for each condition.