rabbit abs to p-ser in pkc substrate motif (6967)  (Cell Signaling Technology Inc)

Journal: Molecular and Cellular Biology

Article Title: Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

doi: 10.1128/MCB.00828-15

Figure Lengend Snippet: Wnt3a and Wnt5a ligands increase the PKC-mediated phosphorylation of GSK-3β in cancer cells and induce the opposite effect in nonmalignant cells. (A and B) Nonmalignant (A) or malignant (B) cells were incubated in the absence or presence of Wnt3a or -5a ligand for 5 min. GSK-3β was immunoprecipitated from the cell lysates, and the immunoprecipitates were analyzed by Western blotting using the anti-P-Ser-PKC substrate antibody. The results shown are representative of at least three independent experiments using different cell preparations. A densitometric analysis was performed to estimate the changes in Wnt-induced GSK-3β phosphorylation levels with respect to the levels found in basal nonstimulated cells. The bar graphs represent the means and SEM from the results of at least three independent assays. (C) Time course of stimulation of RKO cells with Wnt5a (100 ng/ml). GSK-3β was immunoprecipitated from cell lysates obtained at each time point indicated and analyzed by Western blotting using the anti-P-Ser-PKC substrate antibody. IgG is shown as a control for immunoprecipitation equal loading. The results shown are representative of three independent experiments using different cell preparations.

Article Snippet: The rabbit monoclonal antibody against phospho-Ser (P-Ser)-PKC substrate was purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

doi: 10.1128/MCB.00828-15

Figure Lengend Snippet: PKCζ inhibition blocks GSK-3β phosphorylation. (A) Nonmalignant 112CoN cells and malignant RKO or SW480 serum-starved cells were incubated in the absence or presence of the myristoylated (Myr) PKCζ-selective inhibitor (20 μM) for 1 h. Then, the cells were incubated in the absence or presence of Wnt5a (100 ng/ml) for 5 min. GSK-3β was immunoprecipitated from the cell lysates, and the immunoprecipitates were analyzed by Western blotting using the anti-P-Ser-PKC substrate antibody. The results shown are representative of at least three independent experiments using different cell preparations. A densitometric analysis of the changes in Wnt-induced GSK-3β-specific phosphorylation levels with respect to the levels found in basal nonstimulated cells is shown on the right, and the data represent the means and SEM from at least three independent assays. *, P < 0.05; **, P < 0.01. (B) PKCζ knockdown decreases the phosphorylation of GSK-3β. PKCζ-silenced RKO and SW480 cells were serum starved for 6 h and then incubated in the absence or presence of 100 ng/ml Wnt5a for 5 min. GSK-3β was immunoprecipitated from cell extracts, and the immunoprecipitates were analyzed by Western blotting with the anti-P-Ser-PKC substrate antibody. The results shown are representative of at least three independent experiments using different cell preparations. The densitometric analysis shown in the bar graphs represents the means and SEM from at least three independent assays. *, P < 0.05; **, P < 0.01.

Article Snippet: The rabbit monoclonal antibody against phospho-Ser (P-Ser)-PKC substrate was purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot, Knockdown