Journal: Journal of Virology

Article Title: Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

doi: 10.1128/JVI.02001-20

Figure Lengend Snippet: IAV infection induces activation of the EGFR/ERK pathway. (A) A549 cells were infected with WSN or PR8 (MOI = 1) and collected at 0, 5, 10, 15, 30, 60, 90, and 120 min. (B and C) A549 cells were infected with WSN or PR8 (MOI = 1) without (Live) or with treatment at 56°C or 65°C, and then the cells were further cultured for 0, 5, 10, 15, 30, and 60 min. The cell lysates were harvested and subjected to Western blotting with the indicated antibodies.

Article Snippet: The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to EGFR, phospho-EGFR (Y1068), SHP2, phospho-SHP2 (Y542), ERK, phospho-ERK (T202/Y204) (Cell Signaling Technology), β-actin, and Flag (TransGen) at 4°C overnight prior to treatment with horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG at room temperature incubation for 1.5 h. The protein band was developed with NcmECL Ultra Reagent (NCM Biotech) and detected using a FluorChem M imaging system (ProteinSimple).

Techniques: Infection, Activation Assay, Cell Culture, Western Blot

Journal: Journal of Virology

Article Title: Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

doi: 10.1128/JVI.02001-20

Figure Lengend Snippet: Inhibition of the EGFR/ERK pathway reduces IAV replication. (A and B) A549 cells were pretreated with 1 μM afatinib (Afa), 10 μM U0126, or DMSO as a vehicle control for 12 h, followed by WSN, PR8, or CA04 infection (MOI = 1) for 30 min, and the protein samples were harvested. The expression levels of p-EGFR and p-ERK were detected by Western blotting. (C and D) Culture supernatants were harvested at 15 h postinfection and subjected to plaque assay to determine the virus titer. (E) A549 cells were transfected with siRNA corresponding to EGFR (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of siEGFR was determined by Western blotting. (F) After transfection with siEGFR or siCtrl at 80 nM for 24 h, the cells were exposed to WSN (MOI = 1) for 15 h, and then the culture supernatants were collected for plaque assay. Data are means and standard deviations (SD). *, P < 0.05.

Article Snippet: The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to EGFR, phospho-EGFR (Y1068), SHP2, phospho-SHP2 (Y542), ERK, phospho-ERK (T202/Y204) (Cell Signaling Technology), β-actin, and Flag (TransGen) at 4°C overnight prior to treatment with horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG at room temperature incubation for 1.5 h. The protein band was developed with NcmECL Ultra Reagent (NCM Biotech) and detected using a FluorChem M imaging system (ProteinSimple).

Techniques: Inhibition, Control, Infection, Expressing, Western Blot, Plaque Assay, Virus, Transfection, Knockdown

Journal: Journal of Virology

Article Title: Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

doi: 10.1128/JVI.02001-20

Figure Lengend Snippet: Inhibition of IAV-induced EGFR/ERK signaling upregulates the expression of innate immunity-related genes. A549 cells were pretreated with 1 μM afatinib (Afa) for 12 h, followed by infection with WSN (A and C), PR8 (E), or CA04 (F) (MOI = 1). (B and D) After U0126 treatment at a concentration of 10 μM for 12 h, A549 cells were infected with WSN (MOI = 1). Total RNA was extracted at 8 h postinfection, and the mRNA levels of IFNs and ISGs were determined by qRT-PCR. (G and H) A549 cells were treated with 1 μM Afa or 10 μM U0126 prior to WSN infection (MOI = 1). IFN-β and IL-29 levels in the culture supernatants collected at 15 h postinfection were measured by ELISA. Data are means and SD. *, P < 0.05; **, P < 0.01.

Article Snippet: The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to EGFR, phospho-EGFR (Y1068), SHP2, phospho-SHP2 (Y542), ERK, phospho-ERK (T202/Y204) (Cell Signaling Technology), β-actin, and Flag (TransGen) at 4°C overnight prior to treatment with horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG at room temperature incubation for 1.5 h. The protein band was developed with NcmECL Ultra Reagent (NCM Biotech) and detected using a FluorChem M imaging system (ProteinSimple).

Techniques: Inhibition, Expressing, Infection, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

doi: 10.1128/JVI.02001-20

Figure Lengend Snippet: Knockdown of EGFR upregulates the expression of innate immunity-related genes during IAV infection. A549 cells were transfected with siEGFR or scrambled control siRNA (siCtrl) at 80 nM for 24 h, followed by infection with WSN (MOI = 1). At 8 h postinfection, total RNA was extracted, and the mRNA levels of IFNs (A) and ISGs (B) were determined by qRT-PCR. (C) IFN-β and IL-29 levels in the culture supernatants collected at 15 h postinfection were measured by ELISA. Data are means and SD. **, P < 0.01.

Article Snippet: The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to EGFR, phospho-EGFR (Y1068), SHP2, phospho-SHP2 (Y542), ERK, phospho-ERK (T202/Y204) (Cell Signaling Technology), β-actin, and Flag (TransGen) at 4°C overnight prior to treatment with horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG at room temperature incubation for 1.5 h. The protein band was developed with NcmECL Ultra Reagent (NCM Biotech) and detected using a FluorChem M imaging system (ProteinSimple).

Techniques: Knockdown, Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Protein Tyrosine Phosphatase SHP2 Suppresses Host Innate Immunity against Influenza A Virus by Regulating EGFR-Mediated Signaling

doi: 10.1128/JVI.02001-20

Figure Lengend Snippet: SHP2 is involved in EGFR-mediated activation of ERK. (A) A549 cells were serum starved for 12 h and then treated with 10 ng/ml EGF for 0, 5, 10, 15, 30, and 60 min. The cell lysates were harvested and subjected to Western blotting with the indicated antibodies. (B and C) After transfection with siRNA corresponding to SHP2 (siSHP2) or scrambled control siRNA (siCtrl) at 120 nM for 24 h, A549 cells were infected with WSN (MOI = 1) or treated with 10 ng/ml EGF for 30 min, and the protein extracts were analyzed by Western blotting with the indicated antibodies. (D and E) A549 cells were pretreated with 1 μM afatinib (Afa), 10 μM SHP099 (SHP), or 10 μM U0126 for 12 h and then infected with WSN (MOI = 1) or stimulated with 10 ng/ml EGF for 30 min. After incubation, the cell lysates were harvested and subjected to Western blotting with the indicated antibodies.

Article Snippet: The membrane was soaked in 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with primary antibodies specific to EGFR, phospho-EGFR (Y1068), SHP2, phospho-SHP2 (Y542), ERK, phospho-ERK (T202/Y204) (Cell Signaling Technology), β-actin, and Flag (TransGen) at 4°C overnight prior to treatment with horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG at room temperature incubation for 1.5 h. The protein band was developed with NcmECL Ultra Reagent (NCM Biotech) and detected using a FluorChem M imaging system (ProteinSimple).

Techniques: Activation Assay, Western Blot, Transfection, Control, Infection, Incubation