Journal: Molecular Neurodegeneration

Article Title: GSK3β-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functions

doi: 10.1186/s13024-018-0295-z

Figure Lengend Snippet: Primary antibody information

Article Snippet: pS9-GSK3β , Rabbit , WB(1:1000) , CST , #9322.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: GSK3β-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functions

doi: 10.1186/s13024-018-0295-z

Figure Lengend Snippet: Reduced tau phosphorylation by intravitreal injection of a GSK3β inhibitor protects RGCs from HFD-induced vision and synapse loss. ( a ) Western blotting for IRS-1, phosphorylated-Akt (S473), total Akt, phosphorylated-GSK3β (Ser9), total GSK3β in total retina lysate. Intensities were quantified and normalized against the level of GAPDH or total proteins (Akt or GSK3β) and expressed as percentage of protein abundance in the retina from HFD groups relative to their age-matched controls. Data are means ± SEM. n = 4 mice per group. * P < 0.05 and ** P < 0.01 vs age-match RD controls. ( b ) A GSK3β specific inhibitor TWS119 or vehicle was injected intravitreally into the right eye (HFD-R-TWS119) or left eye (HFD-L-Veh), respectively, in mice fed with HFD for 20 weeks. Representative VEP waveforms and quantification of differences in peak amplitude (N1-P1) are shown. ( c ) Representative double immunostaining for phopho-tau (pS396- or pT205-Tau; green) with Tau-5 (Red), and for synaptophysin (green) with pT231-Tau (red). Scale bar, 100 μm. ( d ) Representative curvilineal profile of protein immunostaining intensity from GCL to OPL across the image shown in c . Data are means ± SEM. n = 5 eyes (b-d) per group. * P < 0.05 vs contralateral eye injected with vehicle

Article Snippet: pS9-GSK3β , Rabbit , WB(1:1000) , CST , #9322.

Techniques: Injection, Western Blot, Double Immunostaining, Immunostaining

Journal: Molecular Neurodegeneration

Article Title: GSK3β-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functions

doi: 10.1186/s13024-018-0295-z

Figure Lengend Snippet: Reduced tau microtubule binding and microtubule stability are associated with synapse loss in primary RGCs upon glucolipotoxicity in a GSK3β-dependent manner. ( a ) Representative images of triple immunostaining for RGC-characteristic marker Thy1 (red), neuronal markers TUJ1 (green) and Map2 (blue). Scale bar, 100 μm. Primary RGCs were then exposed to conditioned medium (HG + PA) for 24 h, in the absence or presence of TWS119. ( b ) Representative images of subcellular expression of pT231-Tau (green) and Thy1 (red) by double immunofluorescence. Scale bar, 20 μm. ( c ) Representative synaptophysin (green; scale bar, 20 μm) immunostaining in RGCs. Areas boxed in are shown at higher magnification in the lower panels. ( d ) Western blotting for synaptophysin from whole cell lysates (Total) or synaptosome fractions (Syn). Intensities were quantified and normalized against the level of GAPDH and expressed as percentages of protein abundance under stimulation relative to control. ( e ) mRNAs of synaptophysin in total lysates or synaptosomes were quantified by Q-PCR. ( f ) Microtubule sedimentation assay. Western blotting for Tau 5 and β-tubulin in the supernatant (SN) and the microtubule pellet (pellet). Relative intensities of each protein in its respective fraction were quantified and normalized against the sum of the intensity value of that protein (total, including both supernatant and pellet fractions). MT, microtubule. ( g ) Western blotting for Ac-tubulin in whole cell lysates. Intensities were normalized against the level of GAPDH. ( h ) Representative images of double immunofluorescence for Ac-tubulin (green) and Tyr-tubulin (red). Scale bar, 40 μm. Data are means ± SEM of three independent experiments. * P < 0.05 and ** P < 0.01 vs control; # P < 0.05 and ## P < 0.01 vs HG + PA

Article Snippet: pS9-GSK3β , Rabbit , WB(1:1000) , CST , #9322.

Techniques: Binding Assay, Triple Immunostaining, Marker, Expressing, Immunofluorescence, Immunostaining, Western Blot, Microtubule Sedimentation Assay

Journal: Molecular Neurodegeneration

Article Title: GSK3β-mediated tau hyperphosphorylation triggers diabetic retinal neurodegeneration by disrupting synaptic and mitochondrial functions

doi: 10.1186/s13024-018-0295-z

Figure Lengend Snippet: Mitochondrial transport and function are impaired at glucolipotoxicity-stressed RGCs in a GSK3β-dependent manner. ( a ) Mitochondrial axonal trafficking was determined by infecting RGCs with an adenovirus vector carrying a foreign gene for mitochondrial complex IV (Ad-GFP-Mito). Representative fluorescence images for GFP-labeled mitochondria within axons (upper panels) and kymograph images of axonal mitochondrial movement (middle and bottom panels) are shown. Traces of moving mitochondria are indicated with white arrow. The average transport speed of movable mitochondria was calculated and expressed as mito velocity (μm/min). ( b ) Activity of mitochondrial complex I and complex IV was measured by spectrophotometry and expressed as nmol/min/mg protein. ( c ) Representative images of double immunofluoresence for Tau 5 (red) and complex I (green). Scale bar, 40 μm. ( d ) Western blotting for complex I and complex IV from whole cell lysates (Total) or synaptosome fractions (Syn). Intensities were quantified and normalized against the level of GAPDH and expressed as percentage of protein abundance under stimulation relative to control. Data are means ± SEM of three ( a , d ) or four ( b ) independent experiments. * P < 0.05 and ** P < 0.01 vs control; # P < 0.05 and ## P < 0.01 vs HG + PA. NS, no significant difference

Article Snippet: pS9-GSK3β , Rabbit , WB(1:1000) , CST , #9322.

Techniques: Plasmid Preparation, Fluorescence, Labeling, Activity Assay, Spectrophotometry, Western Blot

Journal: Cell Death & Disease

Article Title: NF- κ B inhibition reveals a novel role for HGF during skeletal muscle repair

doi: 10.1038/cddis.2015.66

Figure Lengend Snippet: p65 +/− -CM activates an HGF/Met/GSK3 β pathway in RAW cells. ( a ) Western blot demonstrated that activation of RAW cells in PM induced an increase in pS9-GSK3 β within 30 min (left), a response that was amplified by both WT- (middle) and p65 +/− - CM (right). ( b ) Densitometric analysis revealed that when activated in p65 +/− -CM, the fraction of pS9-GSK3 β increased by 3.5-fold in 30 min, an amount significantly higher than WT-CM and PM groups (* P ⩽0.05). ( c ) Inhibition of Met by SU11274 blocked pS9-GSK3 β in RAW cells 30 min after exposure to LPS and p65 +/− -CM ( d ) in a dose-dependent manner (* versus SF+LPS, + versus no LPS, P <0.05) Data are represented as mean±S.E.M. of at least three independent experiments

Article Snippet: Membranes were incubated with monoclonal antibodies (1 : 1000; Cell Signaling, Danvers, MA, USA) to pS9-GSK3 β , total GSK3 β or polyclonal rabbit anti-HGF (1 : 100; Santa Cruz, Dallas, TX, USA) at 4 °C overnight in 5% milk or BSA in TBST.

Techniques: Western Blot, Activation Assay, Amplification, Inhibition

Journal: Cell Death & Disease

Article Title: NF- κ B inhibition reveals a novel role for HGF during skeletal muscle repair

doi: 10.1038/cddis.2015.66

Figure Lengend Snippet: Hgf upregulation correlates with accelerated muscle regeneration in vivo . ( a ) Hgf expression was significantly upregulated in p65 +/− muscle at 3 days after CTX injury (* P ⩽0.05 versus 3 days WT; + P <0.10 versus day 0 WT). ( b ) Representative images from hemotoxylin and eosin staining indicated that compared with WT muscle, the GAS muscle of p65 +/− mice regenerated more rapidly (arrows) following CTX injury. ( c ) Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis of muscle extracts showed a slight increase in total HGF in uninjured p65 +/− muscles. ( d ) pS9-GSK3 β + macrophages were identified in injured skeletal muscle by immunofluorescent costaining for pS9-GSK3 β (green) and CD68 (red). Arrows indicate examples of colocalization and regions outlined with dashes are digitally enlarged and separated by color channel to the right. ( e ) Quantification of the number of pS9-GSK3 β + macrophages per high power field (HPF, x600) indicated that a significantly higher number of pS9-GSK3 β + /CD68 + macrophages were found in p65 +/ − skeletal muscle compared with WT skeletal muscle at 1, 3 and 5 days after injury ( P ⩽0.05). For ( b ), scale bar: 50 μ m, n =6–8 mice per group. For ( d ), scale bar: 20 μ m, n =3 mice per group. Data are displayed as mean±S.E.M.

Article Snippet: Membranes were incubated with monoclonal antibodies (1 : 1000; Cell Signaling, Danvers, MA, USA) to pS9-GSK3 β , total GSK3 β or polyclonal rabbit anti-HGF (1 : 100; Santa Cruz, Dallas, TX, USA) at 4 °C overnight in 5% milk or BSA in TBST.

Techniques: In Vivo, Expressing, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot