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. 2005 Apr 11;169(1):83–91. doi: 10.1083/jcb.200412089

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Copyright © 2005, The Rockefeller University Press

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Figure 4. — Effect of PH domains on the β1γ2-dependent Golgi fragmentation and PKD phosphorylation. (A) β1γ2-mediated Golgi fragmentation is inhibited by specific PH domains in intact cells. HeLa cells were transfected with FLAG-β1HA-γ2 and GFP-PH domains of proteins listed. The organization of the Golgi apparatus was monitored by fluorescence microscopy using anti-TGN46 and -GM130 (late and early Golgi specific markers) antibodies. 200 cells expressing FLAG-β1 were counted to determine the percentage of cells with fragmented Golgi membranes. Percentage of cells transfected and levels of protein expression for each PH domain was similar in all experiments as monitored by immunofluorescence and Western blotting with anti-GFP antibody, respectively. (B and C) Effect of β1γ2 on PKD phosphorylation in the activation loop. HeLa cells were cotransfected with the constructs listed. GST-PKD was immunoprecipitated and analyzed by Western blotting (B) with antibodies against GST,phospho-PKD (Ser916), phospho-PKD (Ser744–748), respectively, and quantitated by densitometric scan (C). Anti-FLAG antibodies were used to monitor the expression level of β1γ2 and β4γ2 in the respective cell extracts (B). Values are means (±SD, vertical bars) of three separate experiments.