Journal: Brain : a journal of neurology

Article Title: Overexpression of Sirtuin 1 protein in neurons prevents and reverses experimental diabetic neuropathy.

doi: 10.1093/brain/awz324

Figure Lengend Snippet: Figure 1 Expression and regulation of SIRT1 in brain and DRGs (n = 6). (A) SIRT1 protein expression was measured by western blot in protein extracts from DRGs using a polyclonal rabbit antibody that recognizes both the endogenous protein and the transgene. In contrast, anti- HA antibody recognizes only the SIRT1 transgene expression. GAPDH was used as loading control. nSIRT1 expression was induced by feeding the bigenic mice with normal diet or suppressed by feeding with DOX-containing diet. (B and C) Coronal hippocampal brain sections from nmito- eYFP (express eYFP in neuronal mitochondria) and nSIRTOE/mito-eYFP mice (express SIRT1 in neurons and eYFP in neuronal mitochondria) were incubated with primary rabbit polyclonal SIRT1 antibody. Sections were then incubated with goat anti-rabbit (Alexa Fluor 594) secondary antibody, followed by Hoechst 33342 counterstain. Brain and DRG sections were imaged on a Keyence BZ-X800E fluorescence microscope using appropriate filters. Optical sectioning function was applied during image requisition to eliminate fluorescence blurring to produce a confocal-like image. (D and E) Sections of DRG from nmito-eYFP and nSIRTOE/mito-eYFP mice were immunostained with primary rabbit polyclonal SIRT1 antibody, then with goat anti-rabbit (Alexa Fluor 594) secondary antibody, and finally with Hoechst 33342 dye. The images represent an overlay of all stains. Higher magnification is shown in the inserts. n = nucleus; m = mitochondrion; s = SIRT1. In DRG, the satellite glial cells that surround DRGs are intensely stained with Hoechst 33342 to mark the nuclei of satellite glial cells. In nSIRT1OE showed clear nuclear localization in hippocampal CA1 neurons and this localization was distinct from mito-eYFP fluorescence. In contrast, in DRG neurons, SIRT1 has a nuclear and cytoplasmic localization. Green = neuronal mitochondria; red = neuronal SIRT1; blue = neuronal nuclei. Scale bars = 50 mm. WT = wild-type.

Article Snippet: The source and dilution of the various antibodies used in this study were: rabbit polyclonal anti-SIRT1 (Millipore 07-131, 1:1000), mouse monoclonal (m)Ab antiDBC1 (Cell Signaling Technology #5857, 1:1000), rabbit mAb anti-PARP (Cell Signaling Technology #9532, 1:1000), goat polyclonal anti-CD38 (Santa Cruz Biotechnology, #SC7049S, 1:1000), rabbit polyclonal acetylated lysine antibody (Cell Signaling Technology, #9441, 1:1000), rabbit anti-seasonal polyclonal HA antibody (Origene #TA160089, 1:1000), rabbit polyclonal PGC-1 antibody (Novus Biologicals, #NBP1-04676, 1:1000), mouse mAb anti b-actin (Cell Signaling Technology, #3700, 1:1000) and rabbit mAb anti GAPDH (Cell Signaling Technology, #5174, 1:1000).

Techniques: Expressing, Western Blot, Control, Incubation, Microscopy, Staining

Journal: Brain : a journal of neurology

Article Title: Overexpression of Sirtuin 1 protein in neurons prevents and reverses experimental diabetic neuropathy.

doi: 10.1093/brain/awz324

Figure Lengend Snippet: Figure 6 NEDD4-1 deacetylation by nSIRT1OE (n = 6). Western blot analysis of NAD +-consuming enzymes and acetylated proteins in DRG protein extracts prepared from wild-type (WT) and nSIRT1OE mice fed either a control diet (CD) or HFD for 4 months. (A) Preparation of DRG protein extracts, blot analysis, the source and the dilution of the antibodies used are described in the ‘Materials and methods’ section. (B–E) Quantification of the intensity of the bands are shown. Significant decrease in SIRT1 protein, detected with the rabbit polyclonal anti-SIRT1 that recognized both endogenous and overexpressed SIRT1 protein (Millipore 07-131, 1:1000) (B), decrease in PGC-1 protein levels (C), increase in cleaved PARP1 protein (D) and increase in acetylated 118 kDa protein (E) were observed in WT + HFD samples, but not in nSIRT1OE DRG neurons with a control diet or HFD. P 5 0.001 WT + HFD compared with nSIRT1OE + HFD, WT + CD, and nSIRT1OE + CD in B, C and E and WT + HFD compared with WT + CD, and nSIRT1OE + CD in D. ###P = 0.008 (D) for a significant decrease in cleaved PARP1 protein in nSIRT1OE + HFD mice when compared with WT + HFD.

Article Snippet: The source and dilution of the various antibodies used in this study were: rabbit polyclonal anti-SIRT1 (Millipore 07-131, 1:1000), mouse monoclonal (m)Ab antiDBC1 (Cell Signaling Technology #5857, 1:1000), rabbit mAb anti-PARP (Cell Signaling Technology #9532, 1:1000), goat polyclonal anti-CD38 (Santa Cruz Biotechnology, #SC7049S, 1:1000), rabbit polyclonal acetylated lysine antibody (Cell Signaling Technology, #9441, 1:1000), rabbit anti-seasonal polyclonal HA antibody (Origene #TA160089, 1:1000), rabbit polyclonal PGC-1 antibody (Novus Biologicals, #NBP1-04676, 1:1000), mouse mAb anti b-actin (Cell Signaling Technology, #3700, 1:1000) and rabbit mAb anti GAPDH (Cell Signaling Technology, #5174, 1:1000).

Techniques: Western Blot, Control

Journal: PLoS Pathogens

Article Title: Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

doi: 10.1371/journal.ppat.1005001

Figure Lengend Snippet: 293T cells were transfected with a reporter plasmid cocktail of NFAT (A and E), NF-κB (B and F) and AP-1 (C), and increasing amount of a plasmid containing either kGPCR or mGPCR. Reporter luciferase assays were performed at 30 hours post-transfection with whole cell lysates to determine activation of NFAT, NF-κB and AP-1. (D) 293T cells were transfected with increasing amount of a plasmid containing kGPCR or mGPCR. Whole cell lysates were analyzed by immunoblotting with anti-HA and anti-actin antibodies. (E and F) NIH 3T3 cells were transfected with NF-κB (E) and NFAT (F) reporter cocktail as in (A), except with lipofectamine. Activation of NF-κB and NFAT was determined by luciferase reporter assays.

Article Snippet: After antigen retrieval, tissue sections were subject to H&E staining and immunohistochemical staining with antibodies against HA epitope (OriGene), CD31 (Abcam), CD3 (Abcam), CD11c (Abcam), Ki67 (Abcam), CD80 (Abcam), IBA-1 (Abcam), rabbit or mouse ABC staining system (Santa Cruz), and DAB substrate kit (Vector laboratories).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activation Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

doi: 10.1371/journal.ppat.1005001

Figure Lengend Snippet: (A) Mouse SVEC endothelial cells stably expressing kGPCR or mGPCR were fixed and stained with antibodies against the HA epitope (kGPCR or mGPCR), TGN46 (Golgi marker) or PDI (ER marker) and nuclei were stained with DAPI. Representative images were collected with Nikon and processed with ImageJ. Scale bar denotes 10 μm. (B) Control SVEC or SVEC stable cells expressing kGPCR or mGPCR were starved overnight and whole cell lysates were analyzed with indicated antibodies. pAKT and tAKT indicate phosphorylated and total AKT, respectively. (C) and (D) Control SVEC or SVEC stable cells expressing kGPCR or mGPCR (5 × 10 5 ), along with 1 × 10 6 bystander regular SVEC cells, were injected into the flank of nude mice. Tumors were photographed at four weeks post-injection (C) and tumor weight was determined (D).

Article Snippet: After antigen retrieval, tissue sections were subject to H&E staining and immunohistochemical staining with antibodies against HA epitope (OriGene), CD31 (Abcam), CD3 (Abcam), CD11c (Abcam), Ki67 (Abcam), CD80 (Abcam), IBA-1 (Abcam), rabbit or mouse ABC staining system (Santa Cruz), and DAB substrate kit (Vector laboratories).

Techniques: Stable Transfection, Expressing, Staining, Marker, Control, Injection

Journal: PLoS Pathogens

Article Title: Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

doi: 10.1371/journal.ppat.1005001

Figure Lengend Snippet: (A) Diagram of the GPCR locus, in relation to the inserted Bla gene (encoding beta-lactamase), of the recombinant γHV68.kGPCR, wherein mGPCR was replaced with kGPCR carrying an upstream HA epitope. (B) and (C) NIH 3T3 cells were infected with recombinant γHV68.wt, γHV68.kGPCR or γHV68 revertant (γHV68.Rev) (MOI = 0.05) and whole cell lysates were prepared with three times of freeze/thaw. Infectious unit of γHV68 was determined by plaque assay using NIH 3T3 monolayer (B) and plaques were photographed (C). (D) and (E) NIH 3T3 were infected with recombinant γHV68 as indicated. Cells were harvested at indicated time points post-infection. Extracted total RNA was analyzed by reverse transcription and real-time PCR with primers specific for indicated genes (D). Whole cell lysates were prepared and analyzed by immunoblotting with antibodies against RTA, thymidine kinase (TK or ORF21) and β-actin (E). For (B) and (D), p values were calculated in reference to the control γHV68 wild-type group, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After antigen retrieval, tissue sections were subject to H&E staining and immunohistochemical staining with antibodies against HA epitope (OriGene), CD31 (Abcam), CD3 (Abcam), CD11c (Abcam), Ki67 (Abcam), CD80 (Abcam), IBA-1 (Abcam), rabbit or mouse ABC staining system (Santa Cruz), and DAB substrate kit (Vector laboratories).

Techniques: Recombinant, Infection, Plaque Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

doi: 10.1371/journal.ppat.1005001

Figure Lengend Snippet: Subcutaneous tumors developed in γHV68.kGPCR-infected BALB/c mice were analyzed by hematoxylin & eosin (H&E) staining (A) and immunohistochemistry staining with antibodies against the HA epitope (kGPCR, B), endothelial marker CD31 (C), proliferation marker Ki67 (D), T cell marker CD3 (E), CD80 (F), macrophage marker IBA-1 (G) and dendritic cell marker CD11c (H). Tumors were also analyzed by immunohistochemistry staining with antibodies against γHV68 LANA (ORF72, latent antigen) (I) and vGAT (ORF75c, lytic antigen) (J). Boxed regions were amplified right below (I) or next (J) to the original images. Scale bars denote 25 μm.

Article Snippet: After antigen retrieval, tissue sections were subject to H&E staining and immunohistochemical staining with antibodies against HA epitope (OriGene), CD31 (Abcam), CD3 (Abcam), CD11c (Abcam), Ki67 (Abcam), CD80 (Abcam), IBA-1 (Abcam), rabbit or mouse ABC staining system (Santa Cruz), and DAB substrate kit (Vector laboratories).

Techniques: Infection, Staining, Immunohistochemistry, Marker, Amplification