orf expression clone for pink1  (Genecopoeia)

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: Primers for Real-time quantitative PCR.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: Virus infection down regulates PINK1 expression in macrophages. (A) Q-PCR analysis of PINK1 mRNA expression in mouse peritoneal macrophages (PMs) (3 × 10 5 ) infected with VSV (MOI = 1), RSV (MOI = 10), or HSV (MOI = 10), respectively, for indicated hours. (B) Immunoblot analysis of PINK1 protein expression in mouse peritoneal macrophages (3 × 10 6 ) infected with VSV for indicated hours. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). (C) Q-PCR analysis of PINK1 mRNA expression in RAW264.7 cells (1 × 10 5 ) infected with VSV for indicated hours. (D) Q-PCR analysis of PINK1 mRNA expression in peripheral blood mononuclear cells (PBMCs) of 38 pediatric patients with RSV infection and 21 healthy children. Data are mean ± SD. Similar results were obtained in three independent experiments.* p < 0.05, ** p < 0.01, compared with control.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

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

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 knockdown or knockout suppresses virus-induced type I interferon and proinflammatory cytokine production. Mouse peritoneal macrophages (PMs) were transfected with 30 nM scrambled negative control siRNA (siNC) or PINK1 siRNA (siPINK1) for 48 h. PINK1 knockout RAW264.7 macrophages (PINK1 −/− ) were generated using CRISPR/Cas9 gene-editing system. (A) Immunoblot analysis of PINK1 expression level in PMs with PINK1 knockdown, or RAW264.7 cells with PINK1 knockout. (B) qPCR analysis of IFN-β mRNA expression in PMs infected for indicated MOIs with VSV for 6 h. (C–E) qPCR and ELISA analysis of IFN-β and IL-6 levels of in PMs infected with VSV, RSV, or HSV, respectively, for indicated hours. (F) qPCR analysis of IFN-β and IL-6 levels in wild type (PINK1 +/+ ) and PINK1 knockout cells (PINK1 −/− ) RAW264.7 cells infected with VSV for indicated hours. Data are mean ± SD and are representative of three independent results. * p < 0.05, ** p < 0.01, compared with control.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Knockdown, Knock-Out, Virus, Transfection, Negative Control, Generated, CRISPR, Western Blot, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 promotes RLR-triggered IRF3 and NF-κB activation in macrophages. Mouse peritoneal macrophages transfected with 30 nM scrambled negative control siRNA (siNC) or PINK1 siRNA (A) , or RAW264.7 cells transfected with plasmids encoding Myc-PINK1 (B) , were infected with VSV for indicated hours. Phosphorylated or total proteins in lysates were detected by western blot. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Activation Assay, Transfection, Negative Control, Infection, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 interacts with TRAF3 and IRF3 upon VSV infection. (A) Mouse peritoneal macrophages were infected with VSV for indicated hours. Immunoblot analysis of endogenous TRAF3, IRF3, Parkin, RIG-I, TBK1, and MAVS immunoprecipitated with antibody to PINK1. IgG was as control. Numbers between two blots indicate densitometry of TRAF3, IRF3, or Parkin relative to that of PINK1 in immunoprecipitates. (B,C) HEK293T cells were transfected with PINK1 expressing plasmid together with Flag-TRAF3 or HA-IRF3 plasmid. Cells were lysated 24 h after transfection for immunoblot analysis of indicated proteins immunoprecipitated with antibody to Flag (B) or HA (C) tag. (D) Confocal microscopy of HEK293T co-transfected with Myc-PINK1 and Flag-TRAF3 plasmids followed by VSV infection for 4 h. MitoTracker (Mito) was used to probe the mitochondrion (red). DAPI served as a marker of nuclei (blue). Scale bar, 5 μm. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Control, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Marker

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 promotes RLR-triggered immune response via kinase domain dependent manner. (A) Immunoblot analysis of HEK293T cells that cotransfected with GFP-TRAF3 plus Flag-PINK1, Flag-PINK1 mutants vectors followed by VSV infection for 4 h, then immunoprecipitated with antibody to Flag tag. (B) Immunoblot analysis of RAW264.7 cells stably overexpressed with empty vector or vector encoding PINK1 and its mutants. (C) qPCR analysis of IFN-β, IL-6, and VSV-G mRNA in stably transfected RAW264.7 cells expression of PINK1 variants, infected with VSV for indicated times. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Western Blot, Infection, Immunoprecipitation, FLAG-tag, Stable Transfection, Plasmid Preparation, Transfection, Expressing

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 inhibits TRAF3 degradation. Mouse peritoneal macrophages transfected with scrambled negative control siRNA (siNC) or PINK1 specific siRNA (30 nM) (A) , or RAW264.7 cells transfected with PINK1 plasmid (B) , were infected with VSV for indicated hours. TRAF3 proteins in lysates were detected by western blot. Numbers below lanes (top) indicate densitometry of the presented protein relative to β-Actin expression in that same lane (below). (C) HEK293T cells were transfected with GFP-TRAF3, HA-Ub, Flag-Parkin, and varying doses of Flag-PINK1 (0, 0.5, and 1 μg) and infected with VSV for 4 h. Cells were treated with MG132 (10 uM) and harvested for immunoblot analysis of K48-Ub and K63-Ub immunoprecipitated with antibody to GFP tag. Data are representative of three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Transfection, Negative Control, Plasmid Preparation, Infection, Western Blot, Expressing, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 associates with YAP1 and inhibits the interaction of YAP1 with IRF3. (A) Mouse peritoneal macrophages were infected with VSV for indicated hours. Immunoblot analysis of endogenous YAP1 immunoprecipitated with antibody to PINK1. IgG was as control. (B) Confocal microscopy of HEK293T cells that co-transfected with Myc-PINK1 and Flag-YAP1 plasmids followed by VSV infection for 4 h. Scale bar, 5 μm. (C) HEK293T cells were transfected for 24 h with plasmids encoding HA-IRF3, Flag-YAP1, and Myc-PINK1. Immunoblot analysis of indicated proteins immunoprecipitated with antibody to Flag tag. (D) Mouse peritoneal macrophages transfected with scrambled negative control siRNA (siNC) or PINK1 specific siRNA (30 nM) were infected with VSV for indicated hours. Phosphorylated or total proteins of YAP1 in lysates were detected by western blot. Similar results were obtained in three independent experiments.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Immunoprecipitation, Control, Confocal Microscopy, Transfection, FLAG-tag, Negative Control

Journal: Frontiers in Immunology

Article Title: Mitochondrial Protein PINK1 Positively Regulates RLR Signaling

doi: 10.3389/fimmu.2019.01069

Figure Lengend Snippet: PINK1 positively regulates the RLR-triggered antiviral immune response by inhibiting TRAF3 degradation and relieving YAP-mediated inhibition of the cellular antiviral response. (1) Virus infection down-regulates PINK1 expression in macrophages. (2) PINK1 promotes RLR-triggered IRF3 activation via inhibiting Parkin-mediated K48-linked ubiquitination of TRAF3, (3) and PINK1 inhibits YAP1/IRF3 complex formation, which ultimately promotes RIG-I triggered antiviral immune response.

Article Snippet: Plasmids expressing PINK1 (RC206970), Parkin (RC221147), and TRAF3 (RC201106) were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Inhibition, Virus, Infection, Expressing, Activation Assay, Ubiquitin Proteomics