Journal: PLoS ONE

Article Title: Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFNγ Induced Gene Expression and STAT1 Phosphorylation

doi: 10.1371/journal.pone.0051448

Figure Lengend Snippet: HFFs were infected with RH(I) or RHΔ rop16 parasites at an MOI ∼7, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed three times with similar results. B. HFFs were infected with RH(I) or RHΔ rop16 parasites for four hours, stimulated with 100 U/ml IFNγ for the last two hours of infection, fixed, and stained with anti-IRF1 (red), anti-phospho-STAT1 Tyr (green), and Hoechst dye (nucleus, blue). Parasites also express GFP (green). Scale bar represents 10 µm. This experiment was performed three times with similar results. C. HFFs were infected with RH(I), RHΔ rop16 , Pru(II), or CEP(III) parasites at an MOI ∼1, or left uninfected, for four hours. Cells were stimulated, or not, with 100 U/ml human IFNγ for the last hour of infection and cell lysates were collected, run on an SDS-PAGE gel, and Western blotted for phospho-STAT1 Ser727 , phospho-STAT1 Tyr701 , total STAT1α, IRF1, GAPDH (host cell loading control) and SAG1 (parasite loading control). This experiment has been performed two times with similar results.

Article Snippet: Antibodies against IRF1 (BD Biosciences #612046), phospho-STAT1 Tyr701 58D6 (Cell Signaling #9167), phospho-STAT1 Ser727 (Cell Signaling #9177), total STAT1α p91 (C-24) (Santa Cruz #345), GAPDH (6C5) (Santa Cruz #32233), and Toxoplasma surface antigen (SAG)-1 (kindly provided by John Boothroyd, Stanford University) were used in immunofluorescence and Western blot assays.

Techniques: Infection, SDS Page, Western Blot, Staining