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

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Ifn-β−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and flow cytometry was conducted at 4hpi to measure surface receptor expression levels of IFNAR1 and IFNAR2. (B and C) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-β. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (D and E) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-γ. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (F) Ifn-β BMDMs were treated with DMSO or increasing amounts of a JAK inhibitor for 1 hour, then stimulated with 200pg/ml IFN-β for 4 hours. Cell lysates were then collected and immunoblotted for pSTAT1 (Y701), total STAT1, and β-actin. Densitometry was performed using ImageJ software and phosphorylated protein bands were normalized to loading control for each condition. Densitometric ratios are relative to the UI +IFNβ or the UI +IFNγ conditions. Data and densities shown represent one representative experiment out of three.

Article Snippet: After infection, BMDMs were blocked with 5% FCS and rat anti-mouse CD16/CD32 Fc Block (BD Biosciences, 553141) for 15 min followed by incubation with either PE-conjugated mouse anti-IFNAR1 (Biolegend, 127311) or PE-conjugated goat anti-IFNAR2 (R&D Systems, FAB1083P) for 30 min on ice.

Techniques: Infection, Flow Cytometry, Expressing, Software