Figure 6. Regulation of FA exchange... | Rockefeller University Press

Figure 6.

$Figure 6. Regulation of FA exchange requires both the Mgat5/Gal-3 lattice and Cav1. (A) Equal protein amounts of cell lysates from Mgat5+/+, Mgat5−/−ESC, and ESC-Rescue cells were blotted with L-PHA-HRP or with antibodies to Gal-3, Cav1, and β-actin and HRP-conjugated secondary antibodies. (B) Mgat5+/+, Mgat5−/−, Rescue, Mgat5−/−ESC, and ESC-Rescue cells as well as ESC-Rescue (ESC-R) cells transfected with control (CTL) or Gal-3 siRNA were surface labeled at 4°C with anti-Gal-3 mAb and analyzed by FACS. The percentage of positive cells is shown (±SEM; n = 4; *, P < 0.001). (C) Cell lysates of Mgat5+/+, Mgat5−/−, Rescue, Mgat5−/−ESC, and ESC-Rescue cells were probed by Western blotting for pFAK(Y397), FAK, and β-actin and HRP-conjugated secondary antibodies. (D) Mgat5+/+, Mgat5−/−ESC, and ESC-Rescue cells were grown in serum-free conditions on fibronectin-coated coverslips (10 μg/ml) for 48 h and stained with Alexa568-Phalloidin (red) and antibodies to paxillin (green). (E) Mgat5+/+, Mgat5−/−, Rescue, Mgat5−/−ESC, and ESC-Rescue cells were grown in serum-free conditions on fibronectin-coated coverslips (10 μg/ml) for 48 h, labeled with Alexa568-Phalloidin, and cell area determined (±SEM; n = 3; *, P < 0.05; **, P < 0.005). (F) Migration of Mgat5+/+ (black), Mgat5−/−ESC (gray), and ESC-Rescue cells (white) was determined over a 24-h period in serum-free medium on an FN substrate (10 μg/ml) using a wound-healing assay (±SEM; *, P < 0.01). FRAP analysis was performed on Mgat5+/+, Mgat5−/−ESC, and ESC-Rescue cells transfected with FAK-GFP (G) or paxillin-GFP (H). Percentage of recovery (box) shows the extent of the FAK-GFP (G) and paxillin-GFP (H) mobile fraction. Half-time for recovery of fluorescence toward the asymptote of paxillin-GFP is also presented (Box) (H) (*, P < 0.05).$

Regulation of FA exchange requires both the Mgat5/Gal-3 lattice and Cav1. (A) Equal protein amounts of cell lysates from Mgat5^+/+, Mgat5^−/−ESC, and ESC-Rescue cells were blotted with L-PHA-HRP or with antibodies to Gal-3, Cav1, and β-actin and HRP-conjugated secondary antibodies. (B) Mgat5^+/+, Mgat5^−/−, Rescue, Mgat5^−/−ESC, and ESC-Rescue cells as well as ESC-Rescue (ESC-R) cells transfected with control (CTL) or Gal-3 siRNA were surface labeled at 4°C with anti-Gal-3 mAb and analyzed by FACS. The percentage of positive cells is shown (±SEM; n = 4; *, P < 0.001). (C) Cell lysates of Mgat5^+/+, Mgat5^−/−, Rescue, Mgat5^−/−ESC, and ESC-Rescue cells were probed by Western blotting for pFAK(Y397), FAK, and β-actin and HRP-conjugated secondary antibodies. (D) Mgat5^+/+, Mgat5^−/−ESC, and ESC-Rescue cells were grown in serum-free conditions on fibronectin-coated coverslips (10 μg/ml) for 48 h and stained with Alexa568-Phalloidin (red) and antibodies to paxillin (green). (E) Mgat5^+/+, Mgat5^−/−, Rescue, Mgat5^−/−ESC, and ESC-Rescue cells were grown in serum-free conditions on fibronectin-coated coverslips (10 μg/ml) for 48 h, labeled with Alexa568-Phalloidin, and cell area determined (±SEM; n = 3; *, P < 0.05; **, P < 0.005). (F) Migration of Mgat5^+/+ (black), Mgat5^−/−ESC (gray), and ESC-Rescue cells (white) was determined over a 24-h period in serum-free medium on an FN substrate (10 μg/ml) using a wound-healing assay (±SEM; *, P < 0.01). FRAP analysis was performed on Mgat5^+/+, Mgat5^−/−ESC, and ESC-Rescue cells transfected with FAK-GFP (G) or paxillin-GFP (H). Percentage of recovery (box) shows the extent of the FAK-GFP (G) and paxillin-GFP (H) mobile fraction. Half-time for recovery of fluorescence toward the asymptote of paxillin-GFP is also presented (Box) (H) (*, P < 0.05).