Figure 4.

Clathrin is required for postmitotic Golgi reformation. A) NRK cells were treated with either nonsilencing (ctrl) or CHC siRNA for 4 d, at which point total cell lysates were prepared and resolved by Western blotting with antibodies to CHC and α-tubulin (loading control). B) siRNA-treated cells (asterisks) were synchronized in mitosis (metaphase, c, d, k, l; telophase, e, f, m, n) and released for 8 h to complete cytokinesis (g, h, o, p), after which they were prepared for immunofluorescence microscopy using antibodies against clathrin (a, c, e, g, i, k, m, o) and GRASP65 (b, d, f, h, j, l, n, p). Scale bar = 10 μm. C) Quantitation of Golgi morphologies. To analyze the effect of depleting CHC levels on Golgi structure, 3 morphological criteria (normal, intermediate, and disconnected) were defined. Number of cells exhibiting each morphological phenotype was determined by counting ∼100 cells.