Journal: Immunology

Article Title: HIV infection suppresses TLR3 activation-mediated antiviral immunity in microglia and macrophages.

doi: 10.1111/imm.13181

Figure Lengend Snippet: Figure 3. Effect of human immunodeficiency virus (HIV) infection on PolyI:C-induced IFN regulating factors (IRFs) and signal transducer and activator of transcription protein (STAT)1/STAT3. HIV-infected microglial cells (HC695), uninfected microglial cells (C20) (a) and HIV-infected (day 4 post-infection) primary human macrophages (b) were treated with PolyI:C for 2 hr. Cellular proteins were then collected for the expres- sion of total and phosphorylated proteins of IRF3, IRF7, STAT1 and STAT3 by Western blot. Representative Western blots shown above are rep- resentative of three independent experiments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal control to normalize the target proteins expression. The relative intensity of each band is calculated based on the control band (untreated), which is defined as 1.

Article Snippet: We demonstrated that HMW PolyI:C is a potent TLR3 agonist to activate intracellular innate immunity.11 The primary antibodies against IRF3, IRF7, phospho-IRF7 (Ser477), signal transducer and activator of transcription protein (STAT)1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), ISG56, GBP5, Viperin, ISG15 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Virus, Infection, Western Blot, Control, Expressing

Journal: DNA and Cell Biology

Article Title: Nucleofection of Expression Vectors Induces a Robust Interferon Response and Inhibition of Cell Proliferation

doi: 10.1089/dna.2012.1950

Figure Lengend Snippet: Activation analysis of transcription factors NfκB and IFN regulatory factor (IRF3). (A, B) Activation of the factors was followed 1 h postnucleofection of NIH3T3 cells with phGfΔG plasmid or after mock nucleofection. Selected confocal microscopy sections are presented. Cells were fixed and stained by (A) an antibody against nuclear factor-kappa (NF-κB) p-65 (green) and by DAPI (blue) or by (B) an IRF3-specific antibody (green) and DAPI (blue). (C) IRF3 phosphorylation in NIH3T3 cells nucleofected with phGfΔG or mock nucleofected was analyzed by Western blotting. Cells were lysed at the indicated times, separated by SDS electrophoresis, and blotted. Immunostaining was performed by phospho-IRF3 (Ser396) antibody, specific for the epitope containing phosphorylated serine at position 396. Polyclonal antibody against the IRF3 protein was used as a control.

Article Snippet: The antibodies used were mouse monoclonal anti-phospho-Histone H2A.X (Ser 139) (Millipore); mouse monoclonal anti β-actin (Sigma-Aldrich); rabbit polyclonal anti-TLR9 (Abcam); rabbit polyclonal anti-IRF3 (Abcam); rabbit monoclonal anti-phospho IRF3 (Ser 396) (Cell Signaling Technology); rabbit polyclonal anti-NF-κB p65 (Santa Cruz); goat anti-mouse and goat anti-rabbit conjugated with peroxidase (Pierce); and goat anti-rabbit IgG conjugated with Alexa fluor 488 (Molecular Probes).

Techniques: Activation Assay, Plasmid Preparation, Confocal Microscopy, Staining, Phospho-proteomics, Western Blot, Electrophoresis, Immunostaining, Control

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

Article Title: Casein kinase II controls TBK1/IRF3 activation in IFN response against viral infection.

doi: 10.4049/jimmunol.1402777

Figure Lengend Snippet: FIGURE 3. CK2 negatively regulates TBK1/IRF3-induced IFN response in TLR3/4 signaling. (A) Murine BMDMs were transducted by lentiviral pLKO.1 expressing either scrambled or ck2a-specific shRNA (sh-ck2a) overnight on day 2 after BMDM differentiation. Five days after puromycin (2 mg/ml) selection, stable pools of BMDMs were treated by either LPS (100 ng/ml) or poly(I:C) (50 mg/ml) for 24 h. Supernatants were collected, and IFN-ab and CXCL10 amounts were measured by ELISA. Data are analyzed by two-way ANOVA and presented as means 6 SEM, and similar results were obtained from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. (B–D) BMDMs stably transducted by sh-scrambled or sh-ck2a were stimulated by LPS (100 ng/ml) (B and C) or poly(I:C) (50 mg/ml) (D) for various times, as indicated. Whole-cell lysates were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes, followed by immunoblotting with respective Abs. All of the experiments have been repeated at least once, and representative data are shown.

Article Snippet: Abs against p-IkBa (Ser32, 2859), p-p38 (Thr180/Tyr182, 9211), p-JNK (Thr183/Tyr185, 4668), JNK (9252), hemagglutinin (HA; 3721), p-TBK1 S172 (5483), TBK1 (3504), p-IRF3 S396 (4947), IRF3 (4302), CK2a (2656), and p-STAT1 (Y701, 7649) were purchased from Cell Signaling Technology.

Techniques: Expressing, shRNA, Selection, Enzyme-linked Immunosorbent Assay, Stable Transfection, SDS Page, Western Blot

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

Article Title: Casein kinase II controls TBK1/IRF3 activation in IFN response against viral infection.

doi: 10.4049/jimmunol.1402777

Figure Lengend Snippet: FIGURE 4. CK2 negatively regulates STING-mediated TBK1 and IRF3 activation in IFN response. (A) sh-scrambled or sh-ck2a–transducted L929 cells were transfected by poly(dA:dT) (1 mg/ml) or poly(I:C) (1 mg/ml) by Lipofectamine 2000 (Invitrogen), following manufacturer’s instruction, or directly treated by DMXAA (100 mg/ml) for 6 h, followed by RNA preparation and RT-PCR. (B and C) sh-scrambled or sh-ck2a–transducted L929 cells (B) and Raw cells (C) were stimulated by DMXAA (100 mg/ml) for various times. Cytosolic and nuclear extracts were prepared as described in Materials and Methods. Five percent of the cytosolic proteins and 20% of the nuclear proteins were resolved by 10% SDS-PAGE. Subsequently, immunoblotting was conducted by indicated Abs. The amounts of Tubulin and Lamin B1 in cytosol versus nuclei detected by respective Abs were used as internal control for fractionation. Data are analyzed by two-way ANOVA and presented as means 6 SEM, and similar results were obtained from three independent experiments. **p , 0.01, ***p , 0.001.

Article Snippet: Abs against p-IkBa (Ser32, 2859), p-p38 (Thr180/Tyr182, 9211), p-JNK (Thr183/Tyr185, 4668), JNK (9252), hemagglutinin (HA; 3721), p-TBK1 S172 (5483), TBK1 (3504), p-IRF3 S396 (4947), IRF3 (4302), CK2a (2656), and p-STAT1 (Y701, 7649) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Control, Fractionation

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

Article Title: Casein kinase II controls TBK1/IRF3 activation in IFN response against viral infection.

doi: 10.4049/jimmunol.1402777

Figure Lengend Snippet: FIGURE 5. CK2 regulates IRF3 phosphorylation through phosphatase PP2A. (A) Plasmids expressing ISRE-firefly luciferase or Renilla luciferase were cotransfected with plasmid expression Trif, MAVS, STING, TBK1, IRF3, IRF3 S396D, or IRF7 along with plasmid expressing CK2a into HEK293T cells by calcium-phosphate method. Transfected cells were harvested 36 h later, and luciferase activities were measured by dual-luciferase assay kit (Promega), according to manufacturer’s instruction. The data are presented as the relative firefly luciferase/Renilla luciferase ratio. (B) L929 cells were either untreated or treated by DMXAA (100 mg/ml) for 3 h. Cell lysates were immunoprecipitated by anti-IgG or anti-CK2a, respectively. Immunoprecipitates were probed by anti–PP2-Ca and anti-CK2a sequentially. (C) sh-scrambled or sh-ppp2ca–transducted L929 cells were either untreated or treated by DMXAA (100 mg/ml) for 3 h. Whole-cell lysates were prepared and immunoprecipitated by anti-IgG or anti-CK2a. Immunoprecipitates absorbed on protein A beads were used for in vitro PP2A phosphatase assay using phospho-peptide KRpTIRR as substrate (EMD Millipore), according to manufacturer’s instruction. Western blots were carried out to examine the phosphorylation of TBK1 and IRF3, respectively. (D) sh-scrambled or sh-ppp2ca– transducted L929 cells were stimulated by DMXAA for 6 h. Whole-cell lysates were harvested and resolved by 8% SDS-PAGE, and were then transferred onto a polyvinylidene difluoride membrane. Western blots were carried out to examine the phosphorylation of TBK1 and IRF3, as well as the expression levels of TBK1, IRF3, and PP2-Ca, respectively. (E) Cell lysates from Raw cells expressing either pIP-Flag, pIP-Flag-PP2A, or pIP-Flag-PP2Amut (L199P, phosphatase dead) were bound to anti-Flag agarose beads for 3 h at 4˚C to purify recombinant PP2A proteins. p-IRF3 proteins were purified from Raw264.7 cells transfected with pIP-HA-IRF3 and stimulated with LPS (100 ng/ml) for 1 h using anti-HA agarose beads. In vitro dephosphorylation assay was performed by incubating equal volumes of purified p-IRF3 with Flag-PP2A or Flag- PP2Amut for 2 h at 37˚C in phosphatase assay buffer, as described previously (69). Following the termination of dephosphorylation assay, p-IRF3 was measured by Western blotting. Above experiments were repeated at least once, and the representative data are shown. *p , 0.05, ***p , 0.001.

Article Snippet: Abs against p-IkBa (Ser32, 2859), p-p38 (Thr180/Tyr182, 9211), p-JNK (Thr183/Tyr185, 4668), JNK (9252), hemagglutinin (HA; 3721), p-TBK1 S172 (5483), TBK1 (3504), p-IRF3 S396 (4947), IRF3 (4302), CK2a (2656), and p-STAT1 (Y701, 7649) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Expressing, Luciferase, Plasmid Preparation, Transfection, Immunoprecipitation, In Vitro, Phosphatase Assay, Western Blot, SDS Page, Membrane, Recombinant, De-Phosphorylation Assay

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

Article Title: Casein kinase II controls TBK1/IRF3 activation in IFN response against viral infection.

doi: 10.4049/jimmunol.1402777

Figure Lengend Snippet: FIGURE 7. Blockade of CK2 kinase activity promotes TBK1 activation and IFN response. (A) L929 cells were untreated or treated by TBB (20 or 100 mM) for 6 and 12 h, respectively. Whole-cell lysates were prepared, and immunoblotting was conducted to detect the phosphorylation of NF-kB p65 and TBK1, respectively. (B) L929 cells were untreated or treated by TBB (100 mM) for 6 h. Cell lysates were immunoprecipitated by control IgG or anti-IRF3, respectively. Immunoprecipitates were probed by anti–p-IRF3 and IRF3 sequentially. (C) L929 cells (1 3 106/ml) were untreated or treated by TBB (20, 50, or 100 mM) for 12 h, and RNAs were extracted for RT-PCR. (D) L929 cells were pretreated by TBB (100 mM) for 12 h, and then infected by VSV-GFP (multiplicity of infection: 0.1) for 24 h. GFP-positive cells were analyzed by fluorescence microscopy, and supernatants were collected for plaque assay. (E) HEK293T cells (1 3 106/ml) were untreated or treated by 50 mM TBB for 12 and 24 h, respectively. Cytosolic and nuclear extracts were resolved by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Immunoblotting was carried out to detect the phosphorylation of TBK1 and STAT1, respectively. The amounts of GAPDH and histone in cytosol versus nuclei detected by respective Abs were used as internal control for fractionation. (F) HEK 293T cells (1 3 106/ml) were pretreated by 50 mM TBB for 12 h, and RNAs were prepared for RT-PCR. (G) HEK 293T cells (1 3 106/ml) were pretreated by 50 mM TBB for 12 h, followed by VSV-GFP infection (multiplicity of infection: 0.1) for 24 h. Supernatants were collected for plaque assay. Data are analyzed by two-tailed unpaired t test and presented as means 6 SEM. Similar results were obtained from at least two independent experiments. ***p , 0.001.

Article Snippet: Abs against p-IkBa (Ser32, 2859), p-p38 (Thr180/Tyr182, 9211), p-JNK (Thr183/Tyr185, 4668), JNK (9252), hemagglutinin (HA; 3721), p-TBK1 S172 (5483), TBK1 (3504), p-IRF3 S396 (4947), IRF3 (4302), CK2a (2656), and p-STAT1 (Y701, 7649) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Activation Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Control, Reverse Transcription Polymerase Chain Reaction, Infection, Microscopy, Plaque Assay, SDS Page, Membrane, Fractionation, Two Tailed Test

Journal: PLoS ONE

Article Title: Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

doi: 10.1371/journal.pone.0020240

Figure Lengend Snippet: Cells were stimulated with 1.0 µg/ml of P. gingivalis LPS, 0.1 µg/ml of E. coli LPS or 0.1 µg/ml of Pam3CSK4 for 12 hrs. Phosphorylation of IRF3 cellular extracts was analyzed by western blotting. Blot is a representative of three independent experiments.

Article Snippet: Rabbit anti-mouse IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho-IRF3 (Cell Signaling Technology, Danvers, MA), rabbit anti-mouse GAPDH (Santa Cruz Biotechnology), peroxidase labeled anti-rabbit antibody (GE Healthcare, Munich, Germany) and ECL Plus Western Blotting Reagent Pack (Amersham Biosciences, Buckinghamshire, UK) were used for western blotting experiments.

Techniques: Phospho-proteomics, Western Blot