Journal: Journal of Biological Chemistry

Article Title: Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

doi: 10.1074/jbc.m114.595868

Figure Lengend Snippet: FIGURE 8. OspE1-E2 interaction with PDLIM7 modulates PKC signaling. A, bacterial spread in HEK293T cells decreased by treatment with PKC inhibitors Gö6976 and Gö6983. B, increase in phosphorylation of MARCKS upon infection of HeLa cells with wild-type S. flexneri. TPA (10 ng/ml), positive control for PKC activation. C, activation of PKC as measured by phosphorylation of MARCKS upon infection with ospE1 ospE2 mutant complemented with ospE1 or empty vector. HeLa cells stably transfected with an RNAi-resistant PDLIM7 or with vector alone. D, densitometry (from experiments in C). E, activation of PKC as in C upon infection of HeLa cells stably transfected with an RNAi-resistant PDLIM7 by ospE1 ospE2 mutant complemented with ospE1 or ospE111. F, densitometry (from experiments in E). G, inhibition of MARCKS phosphorylation upon treatment with PKC inhibitor Gö6983 during S. flexneri infection of HEK293T cells. H, densitometry (from experiments in G). Within each panel, all lanes are from the same blot. a.u., arbitrary units. Mean S.D., *, p 0.05; **, p 0.05 versus each DMSO control condition. Data represent two or three independent experiments.

Article Snippet: Inhibition of PKC—For chemical inhibition of PCK during plaque assays, Gö6976 or Gö6983 (Tocris Bioscience 2253 and 2285) was added at the time the agarose overlay to 10 M, which is above the IC50 for the PKC isoforms they inhibit.

Techniques: Phospho-proteomics, Infection, Positive Control, Activation Assay, Mutagenesis, Plasmid Preparation, Stable Transfection, Transfection, Inhibition, Control

Journal: Neuron

Article Title: Facilitation of Long-Term Potentiation by Muscarinic M 1 Receptors Is Mediated by Inhibition of SK Channels

doi: 10.1016/j.neuron.2010.11.018

Figure Lengend Snippet: PKC Mediates the M 1 Receptor-Induced Inhibition of SK Channels (A) Inhibition of I AHP by 77-LH-28-1 (10 μM) is reduced by preincubation with the PKC inhibitor Go6976 (100 nM). The scale bars represent 30 pA, 50 ms. (B) Prolongation of EPSPs by 77-LH-28-1 is blocked by preincubation with the PKC inhibitor Go6976 (100 nM). Example of peak normalized voltage traces showing a burst of five EPSPs after preincubation with Go6976 (black) and no prolongation in the presence of 10 μM 77-LH-28-1 (red). The scale bar represents 40 ms. The average normalized decay time constant is not significantly prolonged. (C) Prolongation of EPSPs by 77-LH-28-1 is blocked by inclusion of the PKC inhibitor PKC 19-36 in the patch pipette. Example of peak normalized voltage traces showing a burst of five EPSPs after infusion of (Glu27)PKC 19-36 or PKC 19-36 (black). Prolongation in the presence of 10 μM 77-LH-28-1 (red) only occurs in the presence of (Glu27)PKC 19-36. The scale bar represents 40 ms. The average normalized decay time constant is significantly prolonged by 77-LH-28-1 in the presence of (Glu27)PKC 19-36 and this is significantly reduced by PKC 19-36. (D) Inhibition of I AHP by 77-LH-28-1 (10 μM) is unchanged by preincubation with the CK2 inhibitors TBB (10 μM) or TMCB (10 μM). The scale bars represent 30 pA, 50 ms. (E) Incubation in TMCB (10 μM) or TBB (10 μM) greatly prolonged the EPSP compared to control. Example of peak normalized voltage traces showing a burst of five EPSPs under control conditions (black) and, in separate experiments, after incubation in 10 μM TMCB (red) or 10 μM TBB (green). The scale bar represents 60 ms. The average decay time constant is significantly prolonged by incubation in TMCB or TBB. Input resistance after incubation in TMCB or TBB was unchanged compared to control. (F) Acute application of TMCB (10 μM) or TBB (10 μM) prolonged the average normalized decay time constant of the EPSP. (G) Apamin prolongs the duration of summated EPSPs but does not occlude the action of TBB. Example of peak normalized voltage traces showing a burst of five EPSPs under control conditions (black), in the presence of 100 nM apamin (red), and after addition of 10 μM TBB (green). The scale bar represents 40 ms. The average normalized decay time constant is significantly prolonged by addition of apamin, but there is an additional significant prolongation with 30 min application of TBB. (H) Incubation in TMCB does not block the action of 77-LH-28-1. Example of peak normalized voltage traces showing a burst of five EPSPs after incubation in TMCB (black) and with addition of 10 μM 77-LH-28-1 (red). The scale bar represents 60 ms. The average normalized decay time constant shows a significant change in the presence of 77-LH-28-1. (I) Incubation in TBB blocks the action of 77-LH-28-1. Example of peak normalized voltage traces showing a burst of five EPSPs after incubation in TBB (black) and after addition of 10 μM 77-LH-28-1 (red). The scale bar represents 60 ms. The average normalized decay time constant shows no change in the presence of 77-LH-28-1. The data are plotted as the mean ± SEM.

Article Snippet: CGP55845, D-AP5, LY341495, NBQX, picrotoxin, pirenzepine, oxotremorine-m, TBB, (Glu27)PKC 19-36, and Go6976 were purchased from Tocris.

Techniques: Inhibition, Transferring, Incubation, Control, Blocking Assay