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