Fig. 5. Identification of phosphorylation sites required for efficient nuclear import of GST-V in vivo. (A) 10 ng of GST-V or mutants: 4m (S393A, S421A, S423A, T445A); 3m (S393A, S421A, S423A); and T445A alone, were injected into the cytoplasm of oocytes, with and without co-injection of 12 ng of importin-, and GVs and cytoplasms were recovered after 2, 4 and 6 hours. Extracted protein was immunoblotted using antibody directed against the C-terminal peptide of HDACm. (B) Quantitation of results from upper two panels of (A) normalized to ratios of nuclear:cytoplasmic concentration. GST-V plus importin- (filled circles), GST-V minus importin- (open circles); 4m plus importin- (filled squares), 4m minus importin- (open squares). (C) Quantitation of results from lower two panels of (A). 3m plus importin- (filled circles), 3m minus importin- (open circles); T445A plus importin- (filled squares), T445A minus importin- (open squares). (D) Nuclear import of anti-GST IgG on co-injection with GST, GST-V, 3m and T445A. GVs and cytoplasms were isolated after 4 hours and a slot blot of protein was developed using anti-mouse IgG. (E) Nuclear import of GST-V on co-injection of 5 ng of Ran or of the Ran mutant Q69L. (F) Quantitation of results from (E) showing GST-V plus Ran (filled circles), GST-V plus RanQ69L (open circles).