The frozen biopsy samples were homogenized in radioimmunoprecipitation assay buffer for cell lysis and protein extraction (50 mM Tris, 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 0.25% sodium-deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100; pH 7.4; all chemicals purchased from Roth, Karlsruhe, Germany) containing a protease inhibitor mix (Inhibitor mix M, Serva, Heidelberg, Germany), sonicated, and centrifuged at 16 000 g for 5 min. The supernatant was isolated and protein content determined (bicinchoninic acid assay, Pierce, Bonn, Germany). Enzymatic activities of lactate dehydrogenase (LDH),7 (link) citrate synthase (CS),28 cytochrome c oxidase (COX),12 (link) glutathione peroxidase (GPX),11 (link) catalase,8 (link) superoxide dismutase (SOD), manganese SOD (Mn-SOD),7 (link) and nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H oxidase)9 were measured according to standard protocols, and specific activities were calculated.
Inhibitor mix m
Manufactured by Serva Electrophoresis
Sourced in Germany
Inhibitor mix M is a laboratory reagent used to inhibit the activity of specific enzymes during electrophoresis experiments. The product contains a combination of inhibitors that target various enzymatic pathways. It is designed to maintain the integrity of protein samples during separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Gapdh, by HyTest (2 mentions)
Vision capt, by Vilber (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by HyTest (1 mentions)
Murf1, by Abcam (1 mentions)
Anti phospho sapk jnk, by Cell Signaling Technology (1 mentions)
Anti sapk jnk, by Cell Signaling Technology (1 mentions)
Anti nitrotyrosine, by Abcam (1 mentions)
Anti enos, by Santa Cruz Biotechnology (1 mentions)
Anti gp91phox, by Abcam (1 mentions)
Anti lox 1, by Abcam (1 mentions)
7 protocols using inhibitor mix m
1
Enzymatic Activity Quantification in Biopsy Samples
2
Western Blot Analysis of MuRF1 and MuRF2
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Frozen TA muscle samples were homogenized in relax buffer (90 mmol/L HEPES, 126 mmol/L KCl, 36 mmol/L NaCl, 1 mmol/L MgCl, 50 mmol/L EGTA, 8 mmol/L ATP,10 mmol/L creatine phosphate; pH 7.4) containing a protease inhibitor mix (inhibitor mix M, Serva, Heidelberg,Germany) and sonicated. Protein concentration was determined (bicinchoninic acid assay, Pierce, Bonn,Germany), and aliquots (5 μg) were separated by SDS-polyacrylamide gel electrophoresis (10% gels). Proteins were transferred to a polyvinylidene fluoride membrane and incubated overnight at 4 °C with the following primary antibodies: MuRF1 (1/1000, Myomedix Ltd., Neckargemünd, Germany) or MuRF2 (1/1600, Myomedix Ltd., Neckargemünd, Germany). Membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody and specific bands visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc.,Bonn, Germany) and densitometry quantified using a 1D scan software package (Vision-Capt, Vilber Lourmat, Eberhardzell, Germany). Blots were then normalized to the loading control GAPDH (1/30 000; HyTest Ltd., Turku, Finland).
3
Quantifying Protein Expressions in Frozen Tissues
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Frozen tissue samples were homogenized in radioimmunoprecipitation assay buffer containing a mixture of protease inhibitor (inhibitor mix M, Serva, Heidelberg, Germany) and protein expression that was quantified by western blot using specific antibodies to NAD(P)H oxidase (Abcam, Cambridge, UK), MuRF-1 (generous gift of Dr S. Labeit, University Mannheim, Germany), and MAFbx (generated in rabbits against the following peptide sequence CYPRKEQYGDTLQL, Eurogentec, Seraing, Belgium). After incubation with a horseradish peroxidase-conjugated secondary antibody, specific bands were visualized by enzymatic chemiluminescence (Super Signal West Pico, Pierce, Bonn, Germany) and densitometry quantified by a one-dimensional scan software package (Scanalytics, Rockville, USA). Loading differences were controlled by reprobing the blot with an antibody against Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Hytest, Turku, Finland).
4
Gelatine Zymography for MMP-2 Activity
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Matrix metalloproteinase-2 (MMP-2) -activity was measured by gelatine zymography. LV tissue was homogenized in RIPA buffer (20-fold of wet weight) containing protease inhibitors (Inhibitor mix M, SERVA Electrophoresis GmbH, Heidelberg, Germany). Samples were subsequently centrifuged at 16,000× g for 5 min, with the supernatant isolated and protein content determined (BCA assay; Pierce). Equal amounts of protein (10 µg) were mixed with sample buffer (250 mmol/l Tris·HCl pH 7.4, 10% sodium dodecylsulfate, 20% glycerol, and 0.005% bromphenolblue) under nondenaturating conditions. After electrophoresis (10% polyacrylamide gel containing 1% gelatine), gels were washed for 1.5 h in renaturation buffer (2.5% Triton X-100) and incubated in incubation buffer (in mmol/l: 50 Tris pH 7.6, 10 CaCl2, 50 NaCl, and 0.05% Brij-35) at 37 °C for 72 h. Gels were then stained with 0.25% Coomassie Brilliant Blue R-250, and MMP activity was observed as cleared unstained regions. Densitometry was used to quantify activity (OneD scan, Vilber, France).
5
Quantitative Analysis of Muscle Protein Regulators
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For western blot analyses, frozen TA was homogenized in Relax buffer (90 mmol/L HEPES, 126 mmol/L potassium chloride, 36 mmol/L sodium chloride, 1 mmol/L magnesium chloride, 50 mmol/L EGTA, 8 mmol/L ATP, 10 mmol/L creatine phosphate, pH 7.4) containing a protease inhibitor mix (Inhibitor mix M, Serva, Heidelberg, Germany), sonicated, and centrifuged at 16,000xg for 5 min. Protein concentration of the supernatant was determined (BCA assay, Pierce, Bonn, Germany) and aliquots (5–20 μg) were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride membrane (PVDF) and incubated overnight at 4 °C with the following primary antibodies: MuRF1 (1/1000, Abcam, Cambridge, UK), MuRF2 (1:1.600, Myomedix GmbH, Neckargemünd, Germany). Membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody and specific bands visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany) and densitometry quantified using a 1D scan software package (Scanalytics Inc., Rockville, USA). Blots were then normalized to the loading control GAPDH (1/30000; HyTest Ltd, Turku, Finland). All data are presented as fold change relative to control.
6
Protein Quantification in Frozen Muscle
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Frozen muscle samples were homogenized in Relax buffer (90 mMol/L HEPES, 126 mMol/L KCl, 36 mMol/L NaCl, 1 mMol/L MgCl, 50 mMol/L EGTA, 8 mMol/L ATP, 10 mMol/L creatinphosphate; pH 7.4) containing a protease inhibitor mix (Inhibitor mix M, Serva, Heidelberg, Germany). Protein quantification by western blot analysis was performed as recently described21 , 26 using the specific antibodies listed in TableS1 .
7
Protein Extraction and Western Blot Analysis
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Protein was extracted from frozen aortic samples by incubation at 4°C over-night in lysis buffer (50 mmol/L Tris, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 1% NP-40, 0.25% sodium-deoxycholate, 0.1% SDS, 0.1% Triton X-100; pH 7.4), which contained a protease inhibitor mix (Inhibitor mix M, Serva, Heidelberg, Germany). Western blot analyses were performed as recently described 22, 24 using the following antibodies: anti-eNOS (1:200; Santa Cruz), anti p-eNOS-Ser 1177 (1:2000, BD Biosciences, Heidelberg, Germany) anti gp91phox, anti-LOX-1 (both 1:1000, Abcam, Cambridge, UK), antinitrotyrosine (1:1000; Abcam), anti-SAPK/JNK and anti-phospho-SAPK/JNK (both 1:1000; both Cell Signaling, Frankfurt, Germany). Samples were normalized to glyceraldehyde 3-phosphate dehydrogenase (1:30000, HyTest Ltd, Turku, Finland), which served as the loading control.
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