Journal: Cell death & disease

Article Title: Mitochondrial angiotensin receptors in dopaminergic neurons. Role in cell protection and aging-related vulnerability to neurodegeneration.

doi: 10.1038/cddis.2016.327

Figure Lengend Snippet: Figure 1 Specificity of angiotensin receptor antibodies and localization of angiotensin receptors in mitochondria of dopaminergic neurons. (a) The expression of major RAS components in dopaminergic neurons was confirmed by RT-PCR and laser microdissection of dopaminergic neurons retrogradly labeled by intrastriatal injection of fluorescent red retrobeads (RRB). SN section showing labeled dopaminergic neurons before and after laser microdissection for RT-PCR. Expression of TH, AGT, AT1, AT2 and β-actin mRNA in laser-microdissected dopaminergic neurons (right) and homogenates of SN used as a positive control (left) are also shown. (b) Western blot densitometric bands corresponding to 10 μg of AT1 or AT2 receptor overexpression lysate containing a C-terminal DDKepitope tag (DYKDDDDK) fused (left), and 10 μg of empty vector transfected control cell lysate HEK293 (EV, right). A band of 45 kDa was detected with the AT1 antibody, while a band of 50 kDa was detected with the AT2 antibody. A monoclonal antibody against DDK detected the corresponding band in the protein lysates. Colocalization of mitochondria and AT1 and AT2 receptors in primary cultures of the nigral region (c) and neurons from the MES 23.5 dopaminergic cell line (d). Electron microscopy of AT1 and AT2 labeling in a neuronal process (e) and cytoplasm (f) of a dopaminergic neuron. Immunolabeling for AT1 was observed in the outer membrane and cristae (white arrows) of mitochondria. In addition, strong AT1 labeling was also seen in clusters of free ribosomes (empty arrowheads). Immunolabeling for AT2 was present in mitochondrial membranes (white arrows), in the rough endoplasmic reticulum (black arrowheads) and in small clusters of free ribosomes, some of which were in close proximity to labeled mitochondria (empty arrowheads). AGT, angiotensinogen; DA, dopaminergic; MTDR, MitoTracker Deep Red; SN, substantia nigra; TH, tyrosine hydroxylase. Scale bars: (a) 50 and 200 μm (SN section); (c) 5 μm; (d) 10 μm; (e and f) 500 nm

Article Snippet: The specificity of the antibodies used for WB and immunolabeling studies was established in previous studies: AT1 sc-3118135 and AT2 sc-9040.36,37 In addition, the specificity of the antibodies was confirmed in our laboratory by preadsortion with the corresponding synthetic peptide antigen.38 In the present study, we also used WB analysis of lysates from HEK293 cells transfected with AT1 or AT2 tagged to fusion tail DDK (TA50011 from Origene, Rockville, MD, USA; DDK tag: DYKDDDDK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Laser Capture Microdissection, Labeling, Injection, Positive Control, Western Blot, Over Expression, Plasmid Preparation, Transfection, Control, Electron Microscopy, Immunolabeling, Membrane

Journal: Nature communications

Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination.

doi: 10.1038/s41467-019-12145-1

Figure Lengend Snippet: Fig. 4 USP19 acts at the level of TRIF. a Overexpression of USP19 inhibits TRIF-mediated ISRE activation. HEK293 cells were transfected with ISRE reporter and the indicated adapter expression plasmids together with a control or USP19 expression plasmid for 24 h before luciferase assays were performed. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01 (unpaired t-test); N.S., not significant. b USP19 interacts with TRIF in overexpression systems. HEK293 cells were transfected with HA-USP19 and the indicated adapter expression plasmids. Twenty hours after transfection, co- immunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with anti-HA or anti-Flag. c Domain mapping of the interaction between USP19 and TRIF. HEK293 cells were transfected with the indicated truncations before co- immunoprecipitation and immunoblotting analysis with the indicated antibodies. The schematic presentations of USP19 and TRIF truncations are shown at the top. d Effects of USP19 and its truncations on poly(I:C)- and LPS-induced IFN-β promoter activation. The 293-TLR3 or 293-TLR4 cells were transfected with the IFN-β promoter reporter and Flag-USP19 or its truncations for 24 h, and then left untreated, treated with poly(I:C) (50 μg/mL) or LPS (100 ng/ml) respectively for 8 h before luciferase assays. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Dunnett’s test); N.S., not significant. e Endogenous USP19 is associated with TRIF. The 293-TLR3 or 293-TLR4 cells were left untreated or treated with poly (I:C) (100 μg/ml) or LPS (100 ng/ml) respectively for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file. Error bars represent standard deviation of the mean

Article Snippet: Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and D-galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies.

Techniques: Over Expression, Activation Assay, Transfection, Expressing, Control, Plasmid Preparation, Luciferase, Immunoprecipitation, Western Blot, Standard Deviation

Journal: Nature communications

Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination.

doi: 10.1038/s41467-019-12145-1

Figure Lengend Snippet: Fig. 6 USP19-mediated deubiquitination of TRIF impairs its function. a Overexpression of USP19 inhibits the interaction of TRIF with TLR3 but not with TBK1 or TRAF3. HEK293 cells were transfected with Flag-TLR3, Flag-TBK1, or Flag-TRAF3 and HA-TRIF together with a control or Myc-USP19 expression plasmid for 20 h. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with anti-HA or anti-Flag. b Overexpression of USP19 but not USP19(C607S) inhibits TRIF-TLR3 interaction. HEK293 cells were transfected with Flag-TLR3 or Flag-TBK1 and HA-TRIF together with a control or Myc-USP19 expression plasmid for 20 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c USP19-deficiency potentiates TLR3-TRIF association. The control or USP19-deficient 293- TLR3 cells were treated with poly(I:C) (100 μg/ml) or left untreated for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF or control IgG. The immunoprecipitates were analyzed by immunoblots with the indicated antibodies. d Overexpression of USP19 but not USP19(C607S) inhibits TRIF-TLR4 and TRIF-TRAM interactions. HEK293 cells were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. e USP19-deficiency increases TLR4-TRIF association. The control or USP19- deficient 293-TLR4 cells were treated with LPS (100 ng/ml) or left untreated for the indicated times before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and D-galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation