Fig. 6

IQGAP1 interacts and colocalizes with HECTD1 in cell leading edge, and its ubiquitination is regulated by HECTD1. a Lysates of wild-type and Hectd1 ^R/R MEF cells were harvested immediately or plated on FN coated culture dishes for 60 min at 37 °C and were analyzed by anti-IQGAP1 and GAPDH blotting. b Wild-type and Hectd1 ^R/R MEF cells were stained with paxillin (green), IQGAP1 (red) and phalloidin (blue). c IQGAP1 interacts with HECTD1. HEK293 cells were stably transfected with GFP-IQGAP1 and seeded on fibronectin-coated dishes for 60 min, protein lysates were harvested and immunoprecipitated (IP) by GFP antibody. The lP lysates and whole cell lysates were used for detecting HECTD1, PIP5K1A and β-catenin by western blot. CUGBP1 served as a negative control. d Hela cell stably express His-HECTD1 was transiently transfected with GFP-IQGAP1 for 24 h. Cells were starved overnight and plated on FN-coated slides for 60 min, followed by fixation and staining with HECTD1. Note the site of colocalization shown in intensity profiles (white arrows). Pearson’s correlation coefficient was analyzed by Image J software. * P < 0.05. e Endogenous ubiquitination of IQGAP1. Wild-type and Hectd1 ^R/R cells were treated with the proteasome inhibitor MG132 1 μg/ml or DMSO in serum starvation medium for overnight. Cell were lysed immediately or after 60 min seeded on FN coated dishes. The ubiquitination of IQGAP1 was further verified by immunoprecipitating IQGAP1 and detecting with an anti-ubiquitin antibody. f Half-life of IQGAP1 is increased in Hectd1 ^R/R cells. Equal amounts of cells were plated on 100 mm dishes for 24 h and then treated with 100 μg/ml of Cycloheximide (CHX) for further 6 h (hours), 12 h, 24 h and 30 h. Cell pellets were harvested and the expression of IQGAP1 was detected by Western blot. Relative protein expression is quantified by densitometric analysis of Western blots with Image J software, based on three independent experiments