phosphatidylinositol specific phospholipase c pi plc  (Millipore)

Journal: International Journal of Biological Sciences

Article Title: CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

doi: 10.7150/ijbs.7136

Figure Lengend Snippet: CD14 deficiency results in a marginal reduction of B. burgdorferi phagocytosis. (A) RAW264.7 cells were treated with 1 unit of PI-PLC for 20 min at 4 ºC, followed by incubation with Bb914 at an m.o.i = 25. The top panel represents the remaining CD14 surface expression after PI-PLC treatment (red histogram) compared to untreated cells (black histogram). The grey histogram corresponds to the staining with an isotype control antibody. The bottom panel represents internalization of B. burgdorferi . The grey histogram corresponds to a 4 ºC control. (B) CD14-deficient (red histograms) or wild type control BMMs (Black histograms) were assessed by flow cytometry for their capacity to phagocytose Bb914 at m.o.i. = 10 (top panel) and 25 (bottom panel). The grey histogram represents the 4 ºC control. The experiments are representative of at least 3 independent experiments.

Article Snippet: RAW264.7 cells (10 6 /ml) were preincubated for 20 min with 0.5-1 units of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus (Invitrogen) at 4 ºC, followed by extensive washing and incubation with Bb914 at an m.o.i.

Techniques: Incubation, Expressing, Staining, Flow Cytometry

Journal: BMC Molecular Biology

Article Title: The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase

doi: 10.1186/1471-2199-9-86

Figure Lengend Snippet: Treatment of chicken ART4 transfected C-33A cells with PI-PLC and PNGase F . A) C-33A cells (4 × 10 7 /ml) transfected with the chicken ART4 containing plasmid or the empty plasmid (pSecTag B) were stained with the anti-Flag antibody (black bar) or the isotype control (white bar) after being incubated for 1 h at 37°C in the presence or absence of PI-PLC (5 U/ml). Data show the mean of % positive cells ± S.E.M. of three experiments. ** p = 0.007 (Student's t-test) B) Western blot analysis using the anti-Flag antibody was performed with supernatants of chicken ART4 transfected C-33A cells after incubation in the presence or absence of PI-PLC. Data show one representative experiment out of three. C) The supernatant of chicken ART4 transfected cells was incubated for 1 h in the presence or absence of PNGase F (20000 U/ml) at 37°C. PNGase F alone was run as a control. After boiling the samples in SDS-PAGE sample buffer, Western blot analysis using the anti-Flag antibody were carried out. The membrane was stained with Ponceau S (D). Similar results were obtained in three separate experiments.

Article Snippet: C-33A cells (4 × 10 7 /ml) transfected with the chicken ART4 containing plasmid or the empty plasmid (pSecTagB) were suspended in PBS containing 5 U/ml Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) (Invitrogen GmbH, Karlsruhe, Germany) and incubated for 60 min at 37°C.

Techniques: Transfection, Plasmid Preparation, Staining, Incubation, Western Blot, SDS Page