Fig. 5

Loss of HECTD1 impairs formation of focal adhesions. Equal amounts of wild-type and Hectd1 ^R/R MEF cells were seeded on FN coated coverslips for 30, 60 or 90 min. The cells were fixed and co-stained with phalloidin and anti-paxillin (a) or anti-zyxin (b), respectively. Average adhesions/cell and protein intensity were quantified by Lab Image software from 3 independent experiments, according to the sizes (FXs < = 1 μm² [11], FAs > 2–5 μm². paired t test, *P < 0.05). c Wild-type and Hectd1 ^R/R cells were starved for overnight. cell lysates were either harvested immediately as 0 min control, suspended in culture medium at 37 °C for 60 min or plated on FN coated culture dishes for 30, 60 and 90 min at 37 °C. Lysates were analyzed by anti-Src (active) and GAPDH blotting. d After being starved for overnight, lysates of wild-type and Hectd1 ^R/R cells were harvested immediately or plated on FN-coated culture dishes for 60 min at 37 °C. Activity of RhoA was measured by RhoA G-LISA Activation Assay Kit. Activity of RhoA was recorded as fold change from wild-type 0 min group based on three independent experiments (paired t test, *P < 0.05). e Expression of Rac1 and RhoA was determined by Western Blots