Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection.

doi: 10.4049/jimmunol.1003957

Figure Lengend Snippet: FIGURE 3. MKP-1andMKP-3reciprocallyregulateCD40-inducedp38MAPKandERK1/2phosphorylationandtheirassociatedeffectorfunctions.A,Westernblot analysisoflysatesofP388D1cellstransfectedwithMKP-1siRNA,MKP-3siRNA,orcontrolsiRNAfor36handlefteitheruntreatedoranti-CD40Abtreatedfor15min. Quantitative RT-PCR analysis of iNOS2 (B), IL-12 (C), and IL-10 (D) mRNA expression from P388D1 cells transfected with control siRNA, MKP-1 siRNA, or MKP-3 siRNA for 36 h, followed by stimulation with anti-CD40 Ab (3 mg/ml) for 8 h, is shown. mRNA levels of the indicated genes were normalized against GAPDH gene and expressed as relative fold change of the levels in untreated (medium) cells. Data show meanvalues 6SEM. Data from one of the representative experiments are shown.

Article Snippet: MKP-1, MKP-3 and control small interfering RNA (siRNA), transfection medium, and transfection reagent were procured from Santa Cruz Biotechnology. siRNA transfection was carried out according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection.

doi: 10.4049/jimmunol.1003957

Figure Lengend Snippet: FIGURE 6. MKP-1 promotes L. major infection in susceptible BALB/c mice. A, Immunoblot analysis of p38 MAPK, ERK1/2, and MKP-1 from peritoneal macrophages transduced with lentivirally expressed MKP-1 shRNA or control shRNA for 48 h and then either left untreated (medium) or treated with anti-CD40 Ab (3 mg/ml) for 15 min. B, After stimulation with anti-CD40 Ab for 48 h, supernatants from the parallel sets of experiment were harvested for IL-10 and IL-12 ELISA. C, In vitro parasite load and percentage of infectivity in macrophages transduced with Lv-MKP-1 shRNA or control shRNA for 36 h before infection. Cells were then stimulated with anti-CD40 for 60 h, and parasite burden was assessed by Giemsa staining. Data show significant reduction in amastigote multiplication with Lv-MKP-1shRNA plus anti-CD40 Ab (p = 0.01) treatment. D and E, Footpad thickness and parasite load in BALB/c (CD40+/+) and CD40-deficient (CD402/2) mice. Mice were infected with L. major; 2 d later, they were injected with lentivirus-expressing MKP-1 shRNA or control shRNA. Some groups of BALB/c were treated with anti-CD40 Ab (50 mg/mouse) for 3 alternate days beginning on day 4 postinfection; footpad thickness was assessed weekly (D, E, left panels) and parasite load (D, E, right panels) 8 wk postinfection. As compared with the anti-CD40 Ab- treated BALB/c mice, anti-CD40 plus Lv-MKP-1 shRNA-treated BALB/c mice had significantly less footpad swelling and parasite burden (p , 0.001) . Data from one of the representative experiments are shown. The error bars show mean 6 SEM.

Article Snippet: MKP-1, MKP-3 and control small interfering RNA (siRNA), transfection medium, and transfection reagent were procured from Santa Cruz Biotechnology. siRNA transfection was carried out according to the manufacturer’s protocol.

Techniques: Infection, Western Blot, Transduction, shRNA, Control, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Injection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission

doi: 10.1073/pnas.1309078110

Figure Lengend Snippet: TC10 stimulates Cb-dependent gephyrin microcluster formation in COS-7 cells. (A1–A5) Images of COS-7 cells transfected as indicated. GFP-gephyrin accumulates in large cytoplasmic aggregates when expressed alone (A1) or together with Myc-SH3(+)CbII (A3) or HA-TC10 (A4). In the presence of Myc-ΔSH3CbII (A2), GFP-gephyrin forms microclusters at the plasma membrane. Similarly, HA-TC10 and Myc-SH3(+)CbII jointly trigger GFP-gephyrin microcluster formation (A5). (Scale bar, 10 µm.) (B) Schematic representation of the Cb splice variants and mutants, as well as of the TC10 WT and mutants, used in this study. (C1) Percentages of GFP-gephyrin (co)-transfected cells classified as displaying GFP-gephyrin microclusters (>50 puncta per cell; n = 3 independent transfections, n = 321–428 cells). Significance levels compared with cells transfected with GFP-gephyrin only (gray bar) are shown within the bars. (C2) Total numbers of GFP-gephyrin clusters and aggregates from images of transfected COS-7 cells (n = 14–34 transfected cells per transfection condition). Significance indicated as in C1. (C3) Gephyrin puncta counted in C2 were binned according to their size (n = 14–34 cells). (Insets) Relative fractions of small microclusters (0.05–0.2 µm2; Left) and aggregates (>1 µm2; Right). (D) Percentages of microclusters (0.04–0.4 µm2) per cell in COS-7 cells transfected as indicated (n = 6–34 cells). Statistical significance was tested between the conditions without coexpression of HA-TC10 (first four columns) and those in the presence of TC10 (WT, CA, or DN).

Article Snippet: Neurons were transfected at DIV 4 using the CalPhos mammalian transfection kit (Clontech).

Techniques: Transfection

Journal: PLoS ONE

Article Title: PMA and Ionomycin Induce Glioblastoma Cell Death: Activation-Induced Cell-Death-Like Phenomena Occur in Glioma Cells

doi: 10.1371/journal.pone.0076717

Figure Lengend Snippet: The graphs are representative of three independent experiments. (A) The expression of NFAT1 was effectively knocked down in U87 cells by transfection of specific small hairpin RNA. Cell counting (B) and MTT assay (C) showed that the knockdown of NFAT1 prevented the inhibition of proliferation by PMA and Io. (D–G) Cells were stained with PI and analyzed by flow cytometry for cell cycle and death. (D, E) Cell cycle was analyzed by the incorporation of PI. The cells were plated in triplicate and analyzed 24 h after treatment. The percentage of cells in each phase of the cell cycle is indicated in the graphs. Down-regulation of NFAT1 markedly prevented cell cycle arrest induced by PMA and Io. (F, G) Analysis of cell death 48 h after treatment. The percentage of cells in sub-G 0 is shown in the graph. Silence of NFAT1 also prevented PMA and Io-induced U87 cell death. * P <0.05 (compared with the U87-control-shRNA cells).

Article Snippet: The cells were washed twice with 2 mL of shRNA Transfection Medium (Santa Cruz Biotechnology) before 0.8 mL of shRNA Plasmid Transfection Medium was added.

Techniques: Expressing, Transfection, Cell Counting, MTT Assay, Knockdown, Inhibition, Staining, Flow Cytometry, Control, shRNA

Journal: PLoS ONE

Article Title: PMA and Ionomycin Induce Glioblastoma Cell Death: Activation-Induced Cell-Death-Like Phenomena Occur in Glioma Cells

doi: 10.1371/journal.pone.0076717

Figure Lengend Snippet: The graphs are representative of three independent experiments. (A) The expression of NFAT1 was effectively knocked down in U251 cells by transfection of specific small hairpin RNA. (B) The knockdown of NFAT1 prevented the inhibition of proliferation by PMA and Io in U251 cells. (C) The TUNEL assay showed that NFAT1-silencing inhibited the apoptosis induced by PMA and Io. (D) The induction of FasL by PMA/Io was NFAT1-dependent. (E) The TUNEL assay showed that FasL neutralization by a specific anti-FasL antibody significantly inhibited PMA and Io-induced apoptosis in U251 cells. * P <0.05 (compared with the control respectively).

Article Snippet: The cells were washed twice with 2 mL of shRNA Transfection Medium (Santa Cruz Biotechnology) before 0.8 mL of shRNA Plasmid Transfection Medium was added.

Techniques: Expressing, Transfection, Knockdown, Inhibition, TUNEL Assay, Neutralization, Control