FIGURE 7.

A, Nck-1 modulates ELMO1 subcellular localization. HEK293T cells were transfected with Myc-tagged ELMO1 alone, or together with WT Nck-1 or R308K mutant of Nck-1. The localization of ELMO1 and Nck-1 was analyzed by immunofluorescence staining with confocal microscopy. Alexa594-conjugated secondary antibody was used to visualize ELMO1 (red), Alexa 488-conjugated secondary antibody was used to visualize Nck-1 (green). B, Nck-1 promotes ELMO1/Dock180-induced cell migration. Myc-tagged ELMO1 and Flag-tagged Dock180 were transfected into HEK293T cells with WT Nck-1 or R308K mutant of Nck-1, respectively. Boyden chamber motility assay was performed as described under “Experimental Procedures.” Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05 versus vector transfected cells. #, p < 0.05 versus ELMO1 and Dock180 co-transfected cells. C, HEK293T cells were transfected with Nck-1 siRNA alone, or together with siRNA-resistant WT Nck-1, R308K plasmid, respectively. If necessary, empty vector was added to ensure that equal plasmid amounts were transfected in each condition. Boyden chamber motility assay was performed as described under “Experimental Procedures.” Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05 versus scramble siRNA-transfected cells. #, p < 0.05 versus Nck siRNA-transfected cells. Cells from each transfection condition were saved separately, lysed, and immunoblotted to confirm expression of Dock180, ELMO1, WT Nck-1, and R308K mutant of Nck-1.