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Fig. 6. In vitro phosphorylation of the N-terminal region of golgin-160 by MLK3. Bacterially expressed, purified GST or GST-tagged golgin-160 mutants (1-393) or (96-259) were incubated with MLK3 immunoprecipitated from HeLa cells for 20 minutes or 60 minutes in an in vitro kinase assay. (A) (Left) The incorporation of phosphate into GST or GST/golgin-160(1-393) after a 60 minute incubation in the absence or presence of MLK3. (Right) The immunoblot for GST. (B) (Top) The incorporation of phosphate into GST/golgin-160(96-259) after a 20 minute or 60 minute incubation in the absence or presence of MLK3. (Middle, bottom) The immunoblots for GST (middle) and MLK3 proteins (bottom). (C) Purified GST/golgin-160(1-393) was incubated with or without immunoprecipitated MLK3 in the absence or presence of CEP11004 (0.5 µM). Golgin-160 phosphorylation and MLK3 expression are shown in the top two panels. Total GST/golgin-160(1-393) expression by Coomassie staining (CS) or immunoblotting (IB) are shown in the lower two panels.





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