Journal: Scientific Reports
Article Title: ROCK1 is a novel Rac1 effector to regulate tubular endocytic membrane formation during clathrin-independent endocytosis
doi: 10.1038/s41598-017-07130-x
Figure Lengend Snippet: PI(4,5)P 2 -induced tubulation is dynamin-dependent and its inhibition by active Rac1 involves PLC activity. ( a , b ) In the presence or absence of W13 (20 min, 4.5 µg/ml), the percentage of cells with tubules was determined after dynasore treatment (30 min, 150 µM) or dynamin K44A expression (24 h) ( a ) and in cells transfected with a specific dynamin siRNA or a non-targeting siRNA as a control (48 h) ( b ). Downregulation of dynamin expression by its specific siRNA is shown by western blotting. ( c ) The percentage of cells with tubules was determined in cells expressing Cherry-Mem alone or co-expressed with L10-GFP Inp54p or L10-GFP Inp54p D281A , in the presence or absence of W13 (20 min, 4.5 µg/ml). ( d ) The percentage of cells with tubules was determined in 1-hour starved cells expressing Cherry-Mem or Cherry-Rac1 G12V in the presence or absence of PI(4,5)P 2 (20 min, 50 µM), W13 (20 min, 4.5 µg/ml), or GFP-PIP5K expression. ( e , f ) The percentage of cells displaying tubules among the starved cells expressing GFP-mem, GFP-Rac1 G12V , or active Rac1 mutants (F37A and W56A) after the treatment with the PLC-inhibitor U73122 (20 min, 5 µM) ( e ), W13 (20 min, 4.5 µg/ml), or PIP5K overexpression ( f ). Mean values ± SEM from three independent experiments are shown in all cases. Statistical significance between different conditions and the corresponding controls were determined by Student’s t -test, *p < 0.05, **p < 0.01, ***p < 0.001.
Article Snippet: Plasmid encoding dominant negative dynamin (dynamin-K44A) was obtained from ATCC (MBA-93).
Techniques: Inhibition, Activity Assay, Expressing, Transfection, Western Blot, Over Expression