Journal: bioRxiv
Article Title: A Catalytically Inactive Protein Kinase C alpha Mutation Drives Chordoid Glioma by Pathway Rewiring
doi: 10.1101/2025.05.30.657104
Figure Lengend Snippet: A) 20 ng GST-PKCα WT , GST-PKCα D463H , GST-PKCα D463N was subject to in vitro kinase assay against MARCKS peptide substrate for 5 min in the presence or absence of activators DG/PS and Ca 2+ . B) Myc-PKCα WT or Myc-PKCα D463H were isolated by immunoprecipitation from HEK293T cells. Proteins were subject to kinase assay for 30 min across a peptide array of PKCα substrates, incubated with purified PKC, PKC activators DG/PS, Calcium and PMA, and ATP [γ-32P]. Array was imaged by autoradiography. C) Autoradiography intensities of peptide spots from D) normalized to scrambled peptide negative controls, n=3. D) Quantification of normalized autoradiography intensities from D), n=3, bar represents mean intensity across all replicates. (**** p < 0.0001, two-tailed Mann-Whitney test) E) Domain Structure of PKCα. PKCα is a member of the conventional PKC family, with 2 tandem DG-binding C1 domains (orange), and a Ca 2+ -binding C2 domain (yellow). Catalysis is performed by a kinase domain (blue) and regulated by an AGC C-tail and an N-terminal pseudosubstrate (red), which sits in the active site in the absence of agonist. Phosphorylation occurs at the activation loop (pink), turn motif (orange) and hydrophobic motif (green). F) COS7 cells were transfected for 48 h with HA-empty, or HA-PKCα constructs indicated, before lysis and probing for processing phosphosites by western blot. G) Quantification of HA-PKCα upper band intensity, relative to total HA-PKCα. Bars represent mean +/- SEM from n=3 experiments (**** p < 0.0001, 1-way ANOVA and Tukey multiple comparisons test).
Article Snippet: Purified commercial PKCα (SignalChem P61-18G-10) was used as a positive control.
Techniques: In Vitro, Kinase Assay, Isolation, Immunoprecipitation, Peptide Microarray, Incubation, Purification, Autoradiography, Two Tailed Test, MANN-WHITNEY, Binding Assay, Phospho-proteomics, Activation Assay, Transfection, Construct, Lysis, Western Blot
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![A) 20 ng <t>GST-PKCα</t> WT , GST-PKCα D463H , GST-PKCα D463N was subject to in vitro kinase assay against MARCKS peptide substrate for 5 min in the presence or absence of activators DG/PS and Ca 2+ . B) Myc-PKCα WT or Myc-PKCα D463H were isolated by immunoprecipitation from HEK293T cells. Proteins were subject to kinase assay for 30 min across a peptide array of PKCα substrates, incubated <t>with</t> <t>purified</t> PKC, PKC activators DG/PS, Calcium and PMA, and ATP [γ-32P]. Array was imaged by autoradiography. C) Autoradiography intensities of peptide spots from D) normalized to scrambled peptide negative controls, n=3. D) Quantification of normalized autoradiography intensities from D), n=3, bar represents mean intensity across all replicates. (**** p < 0.0001, two-tailed Mann-Whitney test) E) Domain Structure of PKCα. PKCα is a member of the conventional PKC family, with 2 tandem DG-binding C1 domains (orange), and a Ca 2+ -binding C2 domain (yellow). Catalysis is performed by a kinase domain (blue) and regulated by an AGC C-tail and an N-terminal pseudosubstrate (red), which sits in the active site in the absence of agonist. Phosphorylation occurs at the activation loop (pink), turn motif (orange) and hydrophobic motif (green). F) COS7 cells were transfected for 48 h with HA-empty, or HA-PKCα constructs indicated, before lysis and probing for processing phosphosites by western blot. G) Quantification of HA-PKCα upper band intensity, relative to total HA-PKCα. Bars represent mean +/- SEM from n=3 experiments (**** p < 0.0001, 1-way ANOVA and Tukey multiple comparisons test).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_04/10__1101_slash_2025__05__30__657104/10__1101_slash_2025__05__30__657104___F1.large.jpg)
