Journal: eLife

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.7554/eLife.106601

Figure Lengend Snippet: Quantification of SNO-BDH1 ( A ) and total BDH1 ( B ), relative to p97 ATPase loading control and to each other ( C ), in mouse heart (4 hr reperfusion); N = 8 SCoR2 +/+ (+/+), N = 6 SCoR2 -/- (-/-). Representative western blots shown in . Quantification of SNO-BDH1 ( D ) and total BDH1 ( E ), relative to p97 ATPase loading control and to each other ( F ), in mouse liver (1 hr reperfusion); N = 4 each. ( G ) HEK293 cells transfected with empty vector (EV), WT, or C115S BDH1 were treated with 250 μM SNO-CoA or vehicle for 10 min, followed by SNORAC and blotted for SNO-BDH1 and BDH1, together with loading controls SNO-p97 ATPase and input p97 ATPase. Representative of four independent experiments. ( H, I ) CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100 μM ECNO or vehicle, in addition to 100 μg/ml CHX for 6–24 hr. BDH1 protein expression was quantified relative to t=0 (pre-CHX) in ( H ), and area under the curve (AUC) quantified in ( I ); N = 3 samples/group. ( J–M ) Metabolites corresponding to ketolytic energy availability in mouse heart and plasma subjected to sham or I/R injury (1 or 4 hr reperfusion), quantified as ion abundance by LC/MS-based untargeted metabolite profiling: ( J ) plasma acetoacetic acid, ( K ) heart acetoacetic acid, ( L ) heart acetyl-CoA, ( M ) heart phosphocreatine. N = 5 mice per condition per genotype. ( N ) Model depicting effect of SCoR2 deletion on cardiac ketone body metabolism in SCoR2 -/- mice. ( O–R ) Human heart samples (IRB# Pro00005621, N = 13 with diagnosis of non-ischemic cardiomyopathy (NICM) and N = 13 without known cardiac pathophysiology (healthy)) subjected to SNORAC measuring SNO-BDH1 expression relative to loading control (SNO-p97 ATPase), representative SNORAC shown in ( O ), quantification in ( P ). ( Q, R ) SCoR2 protein expression in human heart samples ( N = 7 healthy, N = 10 NICM from same cohort), as determined by western blot relative to p97 ATPase (representative image in ( Q ), quantified in ( R )). ( S–U ) Human heart samples (IRB# Pro00005621, N = 8–9 with diagnosis of ischemic cardiomyopathy (ICM; pink) and N = 10 without known cardiac pathophysiology (healthy; black)) subjected to SNORAC measuring SNO-BDH1 ( S ) or western blot measuring SCoR2 relative to loading control (SNO-p97 ATPase or p97, respectively). Representative SNORAC/western blot gels shown in . Correlation of SNO-BDH1 (normalized to SNO-p97) versus BDH1 (normalized to p97) expression in healthy ( N = 10) and ICM ( N = 8) heart was assessed by simple linear regression in ( U ). Statistical significance in ( A–C ) determined by two-tailed Student’s t -test; ( D–F ) determined by two-tailed Mann–Whitney test; ( H ) determined by two-way ANOVA with Tukey’s multiple comparisons test; ( I ) determined by one-way ANOVA with Tukey’s multiple comparisons test; ( J–L ) determined by multiple independent Student’s t -tests performed between genotypes in each condition; ( M, P ) determined by two-tailed Mann–Whitney test; ( R–T ) determined by Student’s t -test; ( U ) determined by simple linear regression performed to identify SNO-BDH1 versus BDH1 relationship. R 2 and p-value show the goodness of fit of the regression model and significance of slope difference from zero, respectively. *p ≤ 0.05, **p ≤ 0.01, ns = not significant. Figure 4—source data 1. Original western blots for , indicating the relevant bands. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Original western blots for , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. Original western blots for , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Anti-p97 (mouse monoclonal) , Fitzgerald , 10R-P104A , 1:1000 for blotting.

Techniques: Control, Western Blot, Transfection, Plasmid Preparation, Pulse Chase, Expressing, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.7554/eLife.106601

Figure Lengend Snippet: ( A ) Representative gel from SNORAC measuring SNO-BDH1 and western blot measuring total BDH1, relative to p97 ATPase loading control, in mouse heart (4 hr reperfusion), quantified in . ( B ) SNO-BDH1 assessed by SNORAC in SCoR2 +/+ (+/+) and SCoR2 -/- (-/-) liver tissue ( N = 4 each) from mice (1 hr reperfusion), quantified in . ( C ) Representative Western blot, quantified in , denoting a CHX pulse-chase assay, in which HEK293 cells transfected with V5-tagged BDH1 were treated with 100 μg/ml CHX to block new protein synthesis, together with 100 μM ECNO or vehicle, with samples collected at 6–24 hr. Duplicates are shown by treatment condition. ( D, E ) Beta-hydroxybutyrate (β-HB) quantified as ion abundance in +/+ and -/- mouse heart and plasma by LC/MS-based untargeted metabolite profiling, N = 5 mice per condition per genotype. ( F ) Sequences of BDH1, or protein product of closest homology, from selected species aligned with constraint-based multiple alignment tool ( COBALT ); red color shows differences from the H. sapiens BDH1 sequence. Cys 115 is indicated by a black arrow. T. nigroviridis protein product ID is CAG04267 . Representative gel from SNORAC assessing SNO-BDH1 and SNO-PKM2 expression relative to SNO-p97 ATPase loading control ( G ) and from Western blot assessing BDH1, PKM2, and SCoR2 expression relative to p97 ATPase loading control ( H ) in human hearts without ( N = 10) and with ( N = 8–9) diagnosis of ischemic cardiomyopathy (ICM) (IRB # Pro00005621). Statistical significance in ( D, E ) determined by independent Student’s t -tests performed between genotypes at each time point. Figure 4—figure supplement 1—source data 1. Original western blots for (panel A), indicating the relevant bands. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in (panel A). Figure 4—figure supplement 1—source data 3. Original western blots for (panel B), indicating the relevant bands. Figure 4—figure supplement 1—source data 4. Original files for western blot analysis displayed in (panel B). Figure 4—figure supplement 1—source data 5. Original western blots for (panel C), indicating the relevant bands. Figure 4—figure supplement 1—source data 6. Original files for western blot analysis displayed in (panel C). Figure 4—figure supplement 1—source data 7. Original western blots for (panel G), indicating the relevant bands. Figure 4—figure supplement 1—source data 8. Original files for western blot analysis displayed in (panel G). Figure 4—figure supplement 1—source data 9. Original western blots for (panel H), indicating the relevant bands. Figure 4—figure supplement 1—source data 10. Original files for western blot analysis displayed in (panel H).

Article Snippet: Antibody , Anti-p97 (mouse monoclonal) , Fitzgerald , 10R-P104A , 1:1000 for blotting.

Techniques: Western Blot, Control, Pulse Chase, Transfection, Blocking Assay, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Sequencing, Expressing, Biomarker Discovery

Journal: eLife

Article Title: The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming

doi: 10.7554/eLife.106601

Figure Lengend Snippet: ( A ) Human heart samples (IRB# Pro00005621, N = 9 with diagnosis of ischemic cardiomyopathy (ICM) and N = 10 without known cardiac pathophysiology (healthy)) subjected to SNORAC measuring SNO-PKM2 relative to loading control (SNO-p97 ATPase). Representative SNORAC/Western blot gels shown in . ( B–J ) Metabolites quantified by ion abundance in SCoR2 +/+ (+/+) and SCoR2 -/- (-/-) mouse heart and plasma by LC/MS-based untargeted metabolite profiling; N = 5 each condition. ( B ) Heart lactate. ( C–K ) Metabolites organized by relationship to the PPP, as inputs ( C, D ), products ( E–H ), or polyol compounds that are categorized as downstream end products of the PPP ( I, J ). NADPH measured in heart lysate after 2 hr reperfusion, erythrose 4-phosphate measured after 4 hr reperfusion. ( K ) Recombinant SCoR2 activity quantified via NADPH consumption by spectrophotometer in the presence of canonical substrate (100 μM SNO-CoA) or carbohydrates (1 mM); [SCoR2]=186 nM, [NADPH]=100 μM. Results presented as specific activity of SCoR2 (μM substrate consumed/min/mg protein). Assay performed in triplicate. ( L ) Summary model showing SCoR2-mediated regulation of carbohydrate metabolism, including PPP and polyol compounds, to generate NADPH and phosphocreatine in the mouse heart. Green arrows indicate pathway upregulation in the absence of SCoR2, and red arrows indicate downregulation. Blue boxes indicate metabolic pathways, tan boxes indicate metabolites, and pink boxes indicate metabolites of particular significance. Thick black arrows indicate directions of SCoR2-regulated metabolic changes. This panel was created using Biorender.com . Statistical significance in ( A ) determined by Student’s t -test; ( B–H, J ) determined by multiple independent Student’s t -tests performed between genotypes in each condition; ( I ) determined by two-tailed Mann–Whitney test; ( K ) determined by two-tailed Mann–Whitney test between SNO-CoA condition and each carbohydrate condition ( N = 3–8 independent replicates per condition). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Antibody , Anti-p97 (mouse monoclonal) , Fitzgerald , 10R-P104A , 1:1000 for blotting.

Techniques: Biomarker Discovery, Control, Western Blot, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Recombinant, Activity Assay, Spectrophotometry, Two Tailed Test, MANN-WHITNEY

Journal: Brain

Article Title: Reply: Hereditary spastic paraplegia caused by a mutation in the VCP gene VCP: A Jack of all trades in neuro- and myodegeneration?

doi: 10.1093/brain/aws202

Figure Lengend Snippet: Figure 1 VCP and WASH complex expression pattern in a VCP haploinsufficient mouse strain. Left: Note the decreased VCP protein expression (rabbit polyclonal, Novus Biologicals NB100-1557) in conjunction with increased strumpellin protein expression (rabbit poly- clonal EP085378/SY1593) (Clemen et al., 2010) in total protein extract preparations from skeletal muscle tissue derived from wild-type (WT) and VCP haploinsufficient (HEM) littermates. GAPDH was used as an internal loading control (not shown). Right: Quantitative real-time PCR analyses of VCP and WASH complex subunit messenger RNA levels in skeletal muscle tissue derived from the same mice. Note that the messenger RNA levels of 6 out of 7 WASH complex components are upregulated. GAPDH messenger RNA levels were used for normalization and ratios of HEM to WT were calculated. Mean values and standard errors were obtained from two experiments (four measurements each) of two animals per genotype.

Article Snippet: Left: Note the decreased VCP protein expression (rabbit polyclonal, Novus Biologicals NB100-1557) in conjunction with increased strumpellin protein expression (rabbit polyclonal EP085378/SY1593) (Clemen et al., 2010) in total protein extract preparations from skeletal muscle tissue derived from wild-type (WT) and VCP haploinsufficient (HEM) littermates.

Techniques: Expressing, Derivative Assay, Control, Real-time Polymerase Chain Reaction

Journal: Human Molecular Genetics

Article Title: Alteration of the unfolded protein response modifies neurodegeneration in a mouse model of Marinesco–Sjögren syndrome

doi: 10.1093/hmg/ddp464

Figure Lengend Snippet: Ubiquitin-positive protein inclusions in Purkinje cells. ( A – I ) ER and nuclear localization of protein inclusions. Cerebellar sections from 3-month-old Sil1 −/− (A–C), 2-month-old Sil1 −/− ; Hyou1 +/− (D–F) and 3-month-old Sil1 −/− ; Dnajc3 −/− (G–I) mice were immunostained with antibodies against BiP (A, D, G) and ubiquitin (Ub; B, E, H). Merged images are shown in C, F, I. ( J – L ) ERAD chaperone p97/VCP partially co-localizes with ubiquitylated protein inclusions in Sil1 −/− Purkinje cells. Brain sections from 80-day-old Sil1 −/− mice were subjected to immunohistochemistry with antibodies against p97/VCP (J) and ubiquitin (Ub, K). Merged image is shown in L. Scale bar = 5 µm.

Article Snippet: Mouse antibody to p97/VCP (Novus Biologicals) and purified rabbit antiserum to HYOU1 was a used at 1:200 and 1:500 dilutions, respectively ( ).

Techniques: Ubiquitin Proteomics, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of CD11b integrin in the alteration of metabolic factors after phorbol ester stimulation of human myeloid leukemia cells

doi: 10.1186/1478-811X-10-13

Figure Lengend Snippet: Western blot analysis. The pMTH1-U937 (pMTH1) and asCD11b-U937 (asCD11b) cells were cultured in the absence and in the presence of 5nM TPA for 24 h up to 72 h, respectively. Thereafter, the different populations were harvested, lyzed and 40 μg of total cellular protein was separated by 10% SDS-PAGE followed by Western blotting. a ) For the expression of the metabolic enzymes MnSOD, p97/VCP and the 20 S proteasome (α-subunits), gels were subsequently stripped corresponding to a β-actin loading control. b ) Analysis of the expression patterns of different extracellular matrix proteolytic enzymes were tested using antibodies against MMP-1, MMP-7 and MMP-9 for matrix restructuring. The unaltered expression level of β-actin serves as a loading control.

Article Snippet: Following primary antibody incubation (polyclonal anti-MnSOD (Upstate Inc., Lake Placid, NY, USA); polyclonal anti-p97/VCP (Novus Biologicals Inc., Littleton, CO, USA); monoclonal anti-proteasome, clone MCP231 (Enzo Life SciencesGmbH, Lörrach, Germany); polyclonal anti-MMP-1 (Biomol); monoclonal anti-MMP-7, clone 111433 (Biomol); polyclonal anti-MMP-9 (Biomol) and monoclonal anti-β-actin, clone AC-15 (Sigma) for 2 h/37°C, the membranes were washed four times with PBS/Tween and incubated with the appropriate horseraddish peroxidase-conjugated secondary antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h/37°C.

Techniques: Western Blot, Cell Culture, SDS Page, Expressing, Control