Fig. 7. Expression of either of two specific Dyn2 spliced variants, Dyn2(ba) or Dyn2(bb), rescues fluid-phase endocytosis in Dyn2-siRNA-treated cells. (A,A') Fluorescence micrographs of Clone 9 cells treated with transfection reagent alone (Mock) that were allowed to internalize fluorescently conjugated dextran for 60 minutes under low serum conditions and were subsequently immunostained for Dyn2. Mock-treated cells show a punctate plasma membrane and Golgi localization of Dyn2 (A), and a perinuclear accumulation of dextran-positive cytoplasmic vesicles (A'). (B-C') Clone 9 cells were treated with Dyn2 siRNA for 48 hours and then transfected with constructs encoding untagged versions of the different Dyn2 spliced variants. Twenty-four hours later, cells were assayed for dextran internalization and immunostained for Dyn2. Fluorescence micrographs of Dyn2-siRNA-treated cells re-expressing either Dyn2(ba) (B, asterisk) or Dyn2(bb) (C, asterisk) show that dextran uptake has been `rescued' in these cells (B',C'), whereas surrounding cells still depleted of Dyn2 internalize relatively little dextran. (D) Western blot analysis of Dyn2 protein levels in siRNA- and mock-treated cells performed in parallel with the rescue experiments. (E) Quantitation of dextran uptake in cells treated with reagent alone (Mock), Dyn2 siRNA, or Dyn2 siRNA followed by re-expression of the indicated Dyn2 spliced variants based on fluorescence intensity units. All images were acquired and adjusted equally, allowing for a comparison among all conditions. Results represent the average ± s.d. of 50 cells measured in each of two independent experiments.