Journal: PLoS Pathogens

Article Title: RNF26 Temporally Regulates Virus-Triggered Type I Interferon Induction by Two Distinct Mechanisms

doi: 10.1371/journal.ppat.1004358

Figure Lengend Snippet: (A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×10 5 ) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed. (B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×10 6 ) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C). ND, not detected. (D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×10 6 ) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B). (E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×10 6 ) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F). (G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×10 6 ) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. ( n = 3) of a representative experiment performed in triplicate.

Article Snippet: Recombinant IFN-β, TNFα and IL-1β (R&D Systems); mouse monoclonal antibodies against FLAG (Sigma), HA (Covance), β-actin (Sigma), AIF, KDEL (Santa Cruz Biotechnology), β-tubulin (Invitrogen), HSV-1 ICP27, ATG12 (Abcam), TBK1, p-TBK1 and p-IκBα (CST); rabbit polyclonal antibodies against ubiquitin, IRF3, p-IRF3 (Santa Cruz Biotechnology), polyubiquitin K48-linkage and K63-linkage (Millipore) were purchased from the indicated manufacturers.

Techniques: Knockdown, Activation Assay, Transfection, Plasmid Preparation, Infection, Virus, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot

Journal: PLoS Pathogens

Article Title: RNF26 Temporally Regulates Virus-Triggered Type I Interferon Induction by Two Distinct Mechanisms

doi: 10.1371/journal.ppat.1004358

Figure Lengend Snippet: (A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×10 6 ) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×10 6 ) were subjected to quantitative real-time PCR analysis. (B) Effects of RNF26 on IRF3 stability. The 293 cells (1×10 6 ) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26. (C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×10 5 ) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed. (D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×10 6 ) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. (E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×10 6 ) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH 4 Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26. (F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×10 6 ) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. ( n = 3) of a representative experiment performed in triplicate.

Article Snippet: Recombinant IFN-β, TNFα and IL-1β (R&D Systems); mouse monoclonal antibodies against FLAG (Sigma), HA (Covance), β-actin (Sigma), AIF, KDEL (Santa Cruz Biotechnology), β-tubulin (Invitrogen), HSV-1 ICP27, ATG12 (Abcam), TBK1, p-TBK1 and p-IκBα (CST); rabbit polyclonal antibodies against ubiquitin, IRF3, p-IRF3 (Santa Cruz Biotechnology), polyubiquitin K48-linkage and K63-linkage (Millipore) were purchased from the indicated manufacturers.

Techniques: Knockdown, Control, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Activation Assay, Infection, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Regulation of inflammatory signaling by the ST6Gal-I sialyltransferase

doi: 10.1371/journal.pone.0241850

Figure Lengend Snippet: (A) U937 EV and ST6-KD cells were treated with LPS for short-term (15–30 min) or long-term (2–6 hr) timepoints, and lysates were immunoblotted for phosphorylated (activated) or total STAT1 and STAT3. (B) To assess whether TLR4 was α2–6 sialylated, lysates from EV and ST6-KD cells were incubated with agarose-conjugated SNA lectin and sialylated proteins were then precipitated by centrifugation. The precipitates were immunoblotted for TLR4 (upper blot). Total TLR4 expression was evaluated by immunoblotting for TLR4 in whole cell lysates (middle blot). (C) Lysates were probed for phosphorylated or total IRF3, a downstream mediator of LPS signaling. At least two independent experiments were performed for each immunoblot.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in TBS buffer containing 0.1% Tween 20 (TBS-T), and then incubated with antibodies against ST6Gal-I (R&D Systems, AF5924), pNFκB–p65 (S536, Cell Signaling Technology, 3033), total NFκB–p65 (Cell Signaling Technology, 8242), p-STAT1 (Y701, Cell Signaling Technology, 7649), total STAT1 (Cell Signaling Technology, 14994), p-STAT3 (Y705, Cell Signaling Technology, 9145), total STAT3 (Cell Signaling Technology, 9139), p-STAT5 (Y694, Cell Signaling Technology, 4322), total STAT5 (Cell Signaling Technology, 94205), p-IRF3 (Cell Signaling Technology, 37829), total IRF3 (Cell Signaling Technology, 11904) and TLR4 (Novus, NB100-56566).

Techniques: Incubation, Centrifugation, Expressing, Western Blot