pY14Cav1 regulates FAK exchange in MDA-435 human breast cancer cells. (A) MDA-435 cells transfected with vector control (pcDNA), myc-tagged Cav1-mRFP, or Cav1Y14F-mRFP were Western blotted with antibodies to Cav1, pY14Cav1, the myc epitope tag, and β-actin. (B) FRAP analysis of FAK-GFP was performed in MDA-435 cells transfected with FAK-GFP (FAK-GFP only) or cotransfected with FAK-GFP and either Cav1-mRFP or Cav1Y14F-mRFP. A time-lapse sequence (in seconds) shows the corresponding ROI (boxed in red) before photobleaching (prebleach), immediately after photobleaching (bleach), and during recovery (recovery). (C) For MDA-435 cells transfected with FAK-GFP (FAK-GFP only) or cotransfected with FAK-GFP and either Cav1-mRFP or Cav1Y14F-mRFP, percent intensity (±SEM) in the bleached zone of FAK-GFP during recovery and percentage of recovery (box) are shown (n = 3; ±SEM). (D) MDA-435 cells were cotransfected with FAK-GFP and either Cav1-mRFP or CavY14F-mRFP, treated 1 h with PP2, and subjected to FRAP analysis of FAK-GFP. 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). Bar, 20 μm.