Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 2. The pan PKC inhibitor Bis-1 blocked PKC activation and mature OL toxicity induced by SIN-1 and ZnCl2. A, Mature OLs were exposed to a peroxynitrite donor SIN-1 (0.5 mM) or ZnCl2 (100 μM) in the absence or presence of Bis-1 for 30 min, and then lysed for assaying the PKC activity. **, pb0.01 and ***, pb0.001 were obtained when the SIN-1 and the zinc treated groups were compared with the control group. ##, pb0.01 was obtained when the Bis-1 treated groups were compared with SIN-1 alone and ZnCl2 alone groups. A representative experiment of three that were performed is shown. B, OLs were exposed to SIN-1 (0.5 mM) in the absence or presence of Bis-1 for 2 h, and the toxicity was assessed at 24 h. Bis-1 dose-dependently protected against SIN-1 induced toxicity. *, pb0.05 and **, pb0.01 were obtained when the Bis-1 treated SIN-1 groups were compared with the SIN-1 alone group. A representative experiment of six that were performed is shown. C, OLs were exposed to ZnCl2 (100 μM) in the absence or presence of Bis-1 for 2 h, and the toxicity was assessed at 24 h. Bis-1 dose-dependently protected against ZnCl2 induced toxicity. *, pb0.05 and ***, pb0.001 were obtained when the Bis-1 treated ZnCl2 groups were compared with the zinc alone group. A representative experiment of six that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Activation Assay, Activity Assay, Control

Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 4. Expression of various PKC isoforms in mature OLs. A, RNA samples isolated from OLs were analyzed by reverse transcriptase PCR using the primer pairs (see Table 1) specific for rat PKC α, β, γ, η, ε, δ, θ, λ and ζ. The products were run on a 1% agarose gel impregnated with ethidium bromide. The bands were visualized under UV light. A representative experiment of three that were performed is shown. B, Mature OLs were treated with PMA (100 nM) for 24 h and then lysed to detect the expression of various PKC isoforms. The isoform specific antibodies were used to compare the expression of each PKC specific isoform with and without PMA prolonged treatment. β-actin was used as a loading control for lysates from control (CON) and PMA pretreated cells. A representative experiment of three that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Expressing, Isolation, Reverse Transcription, Agarose Gel Electrophoresis, Control

Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 8. Downregulation of PKCθ attenuated OL toxicity induced by zinc. A, OLs were treated with ZnCl2 in the presence of Bis-1, Go6976 (Go) or rottlerin (Ro) for 2 h, and then lysed for western blot. ZnCl2 caused a significant increase of PKCθ phosphorylation, which was completely blocked by rottlerin, but not by Go6976. Bis-1 also slightly attenuated the phosphorylation of PKCθ. There was no phosphorylation of PKCδ following zinc treatment. A representative experiment of three that were performed is shown. B, OLs were infected with Lentiviral PKCθ shRNA particles for 4 days and then lysed for detection of the expression of PKCθ. Transfection with PKCθ shRNA caused a dramatic reduction of the expression of PKCθ, when compared to the GFP shRNA transfected cells. Prolonged treatment with PMA also completely blocked the expression of PKCθ. A representative experiment of three that were performed is shown. C–D, OLs were infected with Lentiviral PKCθ shRNA particles for 4 days and then treated with ZnCl2 (C) or SIN-1 (D) for 2 h. The toxicity was assessed at an additional 24 h. Transfection with GFP shRNA had no effect on zinc or SIN-1 induced toxicity. However, transfection with PKCθ shRNA significantly attenuated cell death induced by ZnCl2 or SIN-1. *, pb0.05 and **, pb0.01 were obtained when PKCθ shRNA transfected groups were compared with GFP shRNA transfected and the control groups following zinc or SIN-1 treatment. A representative experiment of three that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Western Blot, Phospho-proteomics, Infection, shRNA, Expressing, Transfection, Control

Journal: Cell Death & Disease

Article Title: Protein kinase C θ is required for cardiomyocyte survival and cardiac remodeling

doi: 10.1038/cddis.2010.24

Figure Lengend Snippet: PKC θ is expressed in cardiomyocytes and it is rapidly activated after pressure overload. ( A ) Immunolocalization of PKC θ in cryosections of LV from 2-month-old WT mice (a). Lack of PKC θ expression in PKC θ −/− LV is shown in b. The α -MyHC MF20 antibody (green) was used to identify cardiomyocytes (c, WT; d, PKC θ −/− ). (Bar=50 μ m). ( B ) Western blot analysis of PKC isoforms expression in LV from 2-month-old WT and PKC θ −/− mice. Representative experiment is shown ( n =9 per genotype). The α -tubulin level of expression was used for normalization. ( C ) Left panel: representative western blot of the indicated PKC isoforms content in both cytosolic (Cy) and particulate (P) subcellular protein fractions, after 15 min transverse aortic constriction (TAC). Sham-operated mice (Sham) were used as controls. Right panel: densitometric analysis of the results obtained from western blots of the indicated PKC isoforms content in subcellular protein fractions (empty portion of each column=particulate fraction, P; gray portion=cytosolic fraction, Cy) expressed as relative percentage, with 1.0 given as the total level of expression of each isoform. The results were obtained as the mean value (±S.D.) of western blots prepared from protein subcellular fractions of single animals ( n =8 per genotype: 4 sham operated, S, and 4 subjected to TAC, T). ( D ) Western blot analysis of the phosphorylated (active) form of PKC θ ( Thr538 p-PKC θ ) in total protein fractions, after 15 min TAC. The phospho/total PKC θ ratio was evaluated by densitometric analysis and shown in the right. S=sham; T=TAC. * P <0.01

Article Snippet: The following primary antibodies were used: the antimyosin heavy-chain MF20, the anti-ANF (Bachem, Bubendorf, Switzerland); the anti-caveolin-3, the anti-PKC- α , - δ , - ɛ , - η , - θ and the anti-phospho Thr538 PKC θ (BD Bioscience, Franklin Lake, NJ, USA); the anti-Akt, the anti-phosphoAkt, the anti-phosphoGSK3 β and the anti phospho-p38 (Cell Signalling Inc., Danvers, MA, USA); the anti-caspase-9 (Assay Designs, Ann Arbor, MI, USA); the anti-caspase-8 (Santa Cruz Biotec, Santa Cruz, CA, USA); the anti- α -tubulin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot

Journal: Science signaling

Article Title: Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling

doi: 10.1126/scisignal.aar3349

Figure Lengend Snippet: (A) Confocal imaging of Prkcq−/− OT-II CD4+ T cells that were transduced with an RV encoding GFP-tagged WT or HR2A mutant PKCθ (green), mixed with APCs labeled with the cell-tracking dye CMAC (blue), and pulsed with OVA peptide (+OVA). Fixed conjugates were stained for Zap70 plus a secondary fluorescent antibody (AF555). A representative cell is shown. Scale bar, 10 μm.

Article Snippet: For PKCθ, fixed and permeabilized cells were stained with mouse primary antibodies against PKCθ (BD Biosciences) followed by an Alexa Fluor 647-conjugated anti-mouse secondary antibody.

Techniques: Imaging, Transduction, Mutagenesis, Labeling, Cell Tracking Assay, Staining