pY14Cav1 regulates FAK exchange in Mgat5-expressing cells. FRAP analysis was performed on Mgat5^−/−ESC and ESC-Rescue cells transfected with FAK-GFP and either Cav1-mRFP or Cav1Y14F-mRFP (A) or with Cav1-mRFP, Cav1Y14R-mRFP, or Cav1Y14D-mRFP (B) and treated (or not) for 1 h with PP2, as indicated. (C) ESC-Rescue cells were either treated with 20 mM lactose, 20 mM sucrose, or 1 μg/ml SW, or transfected with Gal-3, control (CTL), or Cav1 siRNA for 48 h and then transfected with FAK-GFP (top) or cotransfected with FAK-GFP and Cav1-mRFP (bottom). FRAP analysis of FAK-GFP in FAs was then performed. Percent intensity (±SEM) in the bleached zone of FAK-GFP during recovery and quantification of percentage of recovery (box) are shown (n = 3; ± SEM; *, P < 0.05; **, P < 0.01). (D) Migration of Mgat5^+/+, Mgat5^−/−ESC, and ESC-Rescue cells untreated (black bars) or infected with a Cav1-expressing adenovirus (white bars) was determined over a 24-h period in serum-free medium on an FN substrate (10 μg/ml) using a wound-healing assay. Images show migration of Cav1 adenovirus-infected cells. (E) Mgat5^+/+, Mgat5^−/−ESC, and ESC-Rescue cells were transfected with FAK-GFP alone (left) or FAK-GFP and Cav1-mRFP (right) and FAK-GFP imaged every 30 s for 30 min (see Videos 1–3). Overlay of FAK-GFP images at time 0 (red) and 30 min (green) shows disassembled (red), stable (yellow), and newly formed (green) FAs.