Fig. 4. Inhibition or depletion of Dyn2 reduces internalization of the fluid-phase marker HRP. (A-B') Electron micrographs of HRP uptake in mouse hepatocytes (BNL CL.2) microinjected with control heat-inactivated antibody (A; A', enlargement of boxed area) or the anti-pan-dynamin antibody MC65 (B; B', enlargement of boxed area). Numerous HRP-positive endosomes were detected in control cells following a 60-minute incubation with HRP (A', arrows); however, cells microinjected with native antibody (MC65) displayed very few HRP-positive endocytic structures (B,B'), indicating inhibition of fluid-phase uptake. (C) Quantitation of HRP uptake in mock-treated (black bars) or Dyn2-siRNA-treated (white bars) Clone 9 cells under the indicated serum conditions based on HRP enzyme activity using o-phenylenediamine as a substrate. The ratio of HRP enzyme activity (absorbance at 490 nm) to protein concentration (absorbance at 600 nm) was determined for each sample and is presented as arbitrary units. The amount of internalized HRP was reduced in cells treated with Dyn2 siRNA under all serum conditions when compared with mock-treated cells under similar conditions, although stimulation with 10% FBS or with EGF appeared to induce Dyn2-independent mechanisms of fluid uptake. Results represent the average ± s.d. for three independent experiments.