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. 2000 Oct 24;97(23):12589–12594. doi: 10.1073/pnas.220423497

Figure 5.

Figure 5

Tryptic mapping of phosphorylated GRASP65. GRASP65-FLAG immunoprecipitated from in vitro expression reactions was labeled by purified GST-PlkWT (Upper Right), GST-cyclin B-Cdc2 (Lower Left), and radiolabeled by endogenous kinases (Upper Left) in transfected cells arrested in mitosis by nocodazole treatment and was recovered by immunoprecipitation. Phosphoproteins were eluted from the gel after SDS/PAGE and subjected to tryptic digest. Digestion products were separated in two dimensions by electrophoresis (pH 1.9) and chromatography and detected by autoradiography. Nine major phosphopeptides (labeled 1–9) were detected in a mixture (Lower Right) of in vitro and in vivo products. Plk and Cdc2 phosphorylate specific sites within GRASP65 that are relevant in vivo. The additional phosphopeptide (peptide 7) in the GRASP65 labeled in vivo suggests the existence of at least one more GRASP65 kinase other than Plk and Cdc2.