Figure 1 - GEP100 is responsible for the Matrigel invasion activity of MDA-MB-231 cells.

(a) Expression of ArfGEF mRNAs was analysed by using RT–PCR, coupled with agarose gel electrophoresis. cDNA (2 ng) corresponding to each indicated ArfGEF was used as positive control (PC). NC, without template cDNAs. (b, c) Cells, untransfected or transfected with siRNA duplexes against each indicated ArfGEF, or with irrelevant sequences (Irr), were analysed for their expression of ArfGEFs by immunoblotting of the lysates by using each corresponding ArfGEF antibody, as indicated (b); or were subjected to the Matrigel invasion assay using NIH3T3 conditioned media as chemoattractants (c). Blots of cell lysates (10 g), without siRNA treatment, are also shown (Total, b). (d) Cells treated with a GEP100 siRNA were transfected with a rescue construct of wild-type GEP100 cDNA (resWT) or its Sec7-deletion mutant (resSec7), and analysed for their Matrigel invasion activities as in c. Untreated cells are also included as a control (left column). In c and d, data are presented as percentages calculated by normalizing the values obtained for the untreated cells as 100%. Error bars show mean s.e.m., n = 3. Untreated cells (3597 415, that is, about 3.6% of the initially loaded cells) were calculated to have transmigrated per 6.4 mm Matrigel-coated Boyden chamber filter under these conditions (see also Methods). Uncropped images of blots are shown in Supplementary Fig. 6.