Fig. 4. Reversibility of TGN46 displacement induced by GRIP domain overexpression. (a) HeLa cells transiently transfected with C312 or C312(Y2187A) or control untransfected cells were metabolically labeled with [³⁵S]methionine/cysteine for 30 minutes and then chased for 0, 1, 2, or 4 hours. C312 or C312(Y2187A) was immunoprecipitated from cell lysates at each time point using anti-HA antibodies, fractionated by SDS-PAGE, and total C312 levels were determined by phosphorimaging analysis of the 40 kDa band that was absent in the controls (see Fig. 7). The amount at time 0 was set to 100%, and the percentage remaining at each time point is plotted. A representative of 3 experiments is shown. (b,c) HeLa cells from the same well transiently transfected with C312 were treated with 10 µg/ml CHX for 0, 2 or 4 hours as indicated, fixed, and then analyzed by IFM using antibodies to the HA-epitope (b) and to TGN46 (c). (b) The percentage of cells in a representative experiment that were positively stained with anti-HA (n=238 to 269 per time point in this experiment) were characterized for phenotypic expression level of C312 expression. (c) The percentage of cells in the same experiment that were positively stained with anti-HA were characterized for phenotypic appearance of TGN46 staining; `intact' TGN46 indicates a tight Golgi ribbon as in Fig. 2a.