DENND1A is required for Rab35-dependent Shiga toxin trafficking to the trans-Golgi network. (A) HeLa cells expressing EGFP-tagged DENND1A and the long or short forms of DENND1B were transfected with siRNA duplexes to DENND1A, DENND1B, clathrin heavy chain (CHC), or a nonspecific control for 72 h, then Western blotted as indicated. Molecular mass standards are indicated in kilodaltons. The asterisk indicates a nonspecific cross reaction of the clathrin heavy chain antibody. (B) Dual EGF and STxB uptake assays were performed for 60 min as described previously (Fuchs et al., 2007) in cells transfected with control or DENND1A duplexes for 72 h. Cells were fixed and then stained for the Golgi marker golgin-160. (C) Uptake assays were performed as in B using cells transfected with CHC siRNA duplexes. Cells were fixed and then stained for the transferrin receptor (TfR) to mark recycling endosomes or the TGN marker TGN46. Bars, 10 µm. (D) The extent of EGF and Shiga toxin uptake under the various conditions was measured and is plotted in the graphs (n = 3). ImageJ was used to measure colocalization of markers. Error bars indicate standard error of the mean.