FIG. 6.

eEF2 is phosphorylated during mitosis and dissociates from heavy polysomes. Equal amounts of total protein extracted from cycling nonsynchronized (NS) HeLa cells or cells that were released from a double thymidine block at the indicated times were subjected to immunoblot analysis using antibodies specific for phosphorylated eEF2 (p-eEF2) and for total eEF2 (A) or specific for eEF2K phosphorylated at Ser 366 [p-eEF2K(Ser366)] and for total eEF2K (B). Cycling nonsynchronized (NS) or mitotic (8.5 h after block release; M) cells were used for polysomal analysis. Total protein extracted from each of the 23 sucrose gradient fractions was subjected to immunoblot analysis using antibodies specific for eEF2 (C). The data shown represent one of two independent similar experiments. Band intensities were quantified by densitometry using ImageJ software.