Fig 1: 5' PKC fusions act in a dominant-negative manner, suppressing the activity of wild-type PKC.A, schematic of wild-type and PKCα-CDH8 fusion construct used in this study. In addition to the full-length fusion, a C-deletion construct in which the C-terminal fusion partner is deleted was also generated (PKCα C-Del.). B, analysis of agonist-induced PKC activity in COS7 cells transfected with CKAR alone (Endogenous) or cotransfected with the indicated mCherry-tagged PKC construct. Cells treated with agonists UTP (100 μM), PDBu (200 nM), and inhibitor Gӧ6983 (1 μM). Graphs depict normalized FRET ratio changes (mean ± SEM) from three independent experiments. C, analysis of agonist-induced PKC activity in COS7 cells transfected with the reporter CKAR2 and either vector or the indicated mCherry-tagged PKC construct. Cells treated with UTP (100 μM). Graphs depict normalized FRET ratio changes (mean ± SEM) from three independent experiments. D, analysis of basal PKC activity in COS7 cells transfected with CKAR alone (Endogenous) or cotransfected with the indicated mCherry-tagged PKC construct. Cells treated with inhibitor Gӧ6983 (1 μM). Graphs depict normalized FRET ratio changes (mean ± SEM) from four independent experiments. E, immunoblot analysis of lysates from COS7 cells transfected with the indicated YFP-tagged PKC and treated with PDBu (200 nM) or vehicle (DMSO) for 30 min. Blots probed with antibodies for pSer PKC substrate and GFP. Quantification of pSer PKC substrate signal normalized to loading control (Hsp90) depicts mean ± SEM from three independent experiments. ∗p < 0.05, ∗∗p < 0.01 (Student’s t-test). F, images of cells from (B) prior to drug addition depicting mCherry fluorescence for wild-type PKCα (top) and the PKCα-CDH8 fusion (bottom). Scale bar, 20 μm. G, analysis of agonist-induced PKC activity at the Golgi in COS7 cells transfected with Golgi-targeted CKAR alone (Endogenous) or cotransfected with mCherry-tagged PKCα-CDH8. Cells treated with agonists UTP (100 μM), PDBu (200 nM), and inhibitor Gӧ6983 (1 μM). Graphs depict normalized FRET ratio changes (mean ± SEM) from three independent experiments. H, analysis of basal PKC activity at the Golgi in COS7 cells transfected with Golgi-targeted CKAR alone (Endogenous) or cotransfected with mCherry-tagged PKCα-CDH8. Cells treated with inhibitor Gӧ6983 (250 nM). Graphs depict normalized FRET ratio changes (mean ± SEM) from three independent experiments.
Supplier Page from DNASU for CDH8 (Homo sapiens) in pANT7_cGST (GST-tagged in vitro expression vector)