rabbit polyclonal anti phospho irf7  (Cell Signaling Technology Inc)

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Interaction of the M2-2 protein with IRF7. (A, B, and D) HEK293T cells were transfected with the indicated plasmids. At 24 h posttransfection, the cells were lysed in lysis buffer and subjected to immunoprecipitation (IP) with anti-V5 (A) or anti-FLAG (B and D) antibody, followed by immunoblot analysis (WB) with anti-FLAG, anti-V5, or anti-myc antibody. (C) V5-TRAF6, V5-IKKα, V5-IRF7, and myc-M2-2 were synthesized in the wheat germ cell-free expression system. Then, the in vitro transcription/translation products were mixed in various combinations and subjected to IP with anti-myc antibody followed by WB with anti-V5 and anti-myc antibodies. A portion of the whole-cell lysates (A, B, and D) or the in vitro transcription/translation products (Input) (C) prepared for IP was also subjected to WB. The asterisks indicate the positions of the antibody heavy chain (AbHC).

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Synthesized, Expressing, In Vitro

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Interaction of the M2-2 protein with IRF7 deletion mutants. (A) Schematic diagram of FLAG-tagged deletion mutants of mIRF7. (B and C) HEK293T cells were transfected with the indicated plasmids and lysed at 24 h posttransfection. The lysates were subjected to IP with anti-FLAG antibody followed by WB with anti-myc or anti-FLAG antibody. A portion of the whole-cell lysates prepared for IP was also subjected to WB. (D) HEK293T cells were transfected with the indicated plasmids, an IFN-α6 promoter-driven reporter plasmid, and pRL-TK. At 24 h posttransfection, Fluc and Rluc activities were measured. Relative luciferase activity was calculated as the ratio of Fluc activity to Rluc activity. The mean values from three independent experiments are shown, with standard deviations. The IRF7 deletion mutants used in panel B were of mouse origin, whereas those in panels C and D were of human origin.

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Effect of the M2-2 protein on MyD88-TRAF6-IKKα-induced homodimerization of IRF7. (A and B) HEK293T cells were transfected with the indicated plasmids. At 24 h posttransfection, the cells were suspended in Dulbecco's PBS. After addition of furimazine, luminescence and fluorescence signals were immediately detected. The BRET ratio was calculated as described in Materials and Methods. (C and D) HEK293T cells were transfected with the indicated plasmids and an internal control, pCA7-Fluc. At 36 h posttransfection, Rluc and Fluc activities were measured. Relative luciferase activity was calculated as the ratio of Fluc activity to Rluc activity. The mean values from three independent experiments are shown, with standard deviations.

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Transfection, Fluorescence, Control, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Effect of the M2-2 protein on IPS-1-induced activation and homodimerization of IRF7. (A) HEK293T cells were transfected with the indicated plasmids, pRL-TK, and an IFN-α6 promoter-driven Fluc reporter plasmid. At 24 h posttransfection, Fluc and Rluc activities were measured. Relative luciferase activity was calculated as the ratio of Fluc activity to Rluc activity. (B) HEK293T cells were transfected with the indicated plasmids and pCA7-Fluc. At 36 h posttransfection, Fluc and Rluc activities were measured. Relative luciferase activity was calculated as the ratio of Rluc activity to Fluc activity. The mean values from three independent experiments are shown, with standard deviations. NS3/4A, HCV NS3/4A protein.

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Effect of the M2-2 protein on MyD88-TRAF6-IKKα- or IPS-1-induced phosphorylation of IRF7. (A and B) HEK293T cells were transfected with the indicated plasmids. At 24 h posttransfection, the cells were lysed in Laemmli sample buffer, and the lysates were subjected to WB with the indicated antibodies.

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Phospho-proteomics, Transfection

Journal: Journal of Virology

Article Title: Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

doi: 10.1128/JVI.00579-17

Figure Lengend Snippet: Effect of the M2-2 protein on MyD88-TRAF6-IKKα-induced nuclear translocation of IRF7. (A) HeLa cells cultured in 8-well glass chamber slides were transfected with the indicated plasmids. At 24 h posttransfection, the cells were fixed and permeabilized. Then, the cells were incubated with anti-myc mouse MAb or anti-FLAG rabbit polyclonal antibody as the primary antibody, followed by anti-mouse IgG antibody conjugated to Alexa Fluor 488 (green) or anti-rabbit IgG antibody conjugated to Alexa Fluor 647 (red) as the secondary antibody. The stained cells were mounted with ProLong Gold reagent with DAPI (blue). (B) HEK293T cells were transfected with the indicated plasmids. At 24 h posttransfection, nuclear and cytoplasmic extracts were prepared and then subjected to WB with the indicated antibodies.

Article Snippet: The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz).

Techniques: Translocation Assay, Cell Culture, Transfection, Incubation, Staining