β1-integrin is endocytosed from ruffles in an R-Ras dependent manner. Alexa Fluor555 (red)-labeled β1-integrin antibody was applied to live Cos7 cells expressing: (A-B) GFP-R-Ras-(wt), (C) -(38V), or (D) -(41A). There was significant overlap between β1-integrin and R-Ras (yellow) at ruffles and in vesicles in GFP-R-Ras(wt) and GFP-R-Ras(38V) cells, which was absent in GFP-R-Ras(41A) cells. Note the uniform distribution of β1-integrin in cells expressing GFP-R-Ras(41A) (D). Scale bar is 20 μm. (E-F) Cells were transfected with the following constructs; lanes 1, 6, and 7- GFP-R-Ras(wt), lane 2- GFP-R-Ras(38V); lane 3- GFP-R-Ras(41A), lane 4- GFP, lane 5- untransfected. Following a 30 min incubation with β1-integrin antibody (with the exception of lane 6 as a control, where cells were exposed to antibody for <1 min), cells were biotinylated (with the exception of lane 7, as a control), washed, then lysed and cleared of the biotinylated antibody using strep-avidin beads. The presence of any remaining antibody would be a result of endocytosis during the incubation period, and was quantified by western blot. Densitometry of bands was normalized to lane 7 (wt with no biotinylation) and averaged data (± SEM) from three separate experiments is shown in E. Panel F is data from two experiments where whole cell lysate was probed for β1-integrin as a loading control. Exemplar blots are shown above the bar graphs.
Conklin et al. BMC Cell Biology 2010 11:14 doi:10.1186/1471-2121-11-14