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. Author manuscript; available in PMC: 2008 Apr 1.
Published in final edited form as: Biochim Biophys Acta. 2006 Dec 24;1773(4):483–501. doi: 10.1016/j.bbamcr.2006.12.008

Fig. 6.

Fig. 6

Top: Effect of the PKC inhibitor GF109203X and/or depletion of intracellular calcium with thapsigargin on CCK- (left panel) and TPA- (right panel) stimulated PKD1 S916 and S744/748 phosphorylation in rat pancreatic acini. Rat pancreatic acini were pretreated for with no additions, with GF109203X (5 μM) for 2 h or with thapsigargin (1 μM) in a calcium-free medium (with EGTA 5 μM) for 1 h. Acini were then incubated with no additions (control), with 10 nM CCK for 10 min or with 1 μM TPA for 5 min and then lysed. Upper 3 panels: membranes were analyzed using anti-pS916 PKD1 Ab (panel 1) or anti-pS744/748 PKD1 Ab (panel 2). To verify loading of equal amounts of protein, membranes were stripped and re-blotted with anti-PKD1 Ab (panel 3). Lower panel: For positive control for calcium-free conditions, membranes were analyzed using anti-phospho PYK2 (Y402) Ab (panel 4). The bands were visualized using chemiluminescence and quantification of phosphorylation was assessed using scanning densitometry. The upper part shows a representative experiment of 5 independent experiments. Bottom: Effect of an acute increase in intracellular calcium on PKD1 phosphorylation. Rat pancreatic acini were treated with TPA (10 nM) and/or Thapsigargin (1μM) for 10 min and then lysed. Membranes were analyzed using anti-pS916 PKD1 Ab (panel 1) or anti-pS744/748 PKD1 Ab (panel 2). To verify loading of equal amounts of protein, membranes were stripped and re-blotted with anti-PKD1 Ab (panel 3). A representative blot of 3 independent experiments is shown.