We measured BCKD activity using a modified version of a previous assay (White et al., 2018 (link)). Approximately 30 mg of frozen gastrocnemius muscle or liver were crushed in liquid nitrogen and homogenized (Bio‐Gen PRO200 Homogenizer, PRO Scientific, Oxford, CT) in 250 μL of ice‐cold buffer 1 (30 mM potassium phosphate buffer (KPI), 3 mM EDTA, 5 mM DTT, 1 mM valine, 3% FBS, 5% Triton X‐100 and 1 μm leupeptin (Sigma Aldrich, #L2884)). The resulting sample was centrifuged (10 min at 10,000 g, 4°C) and 50 μL of the supernatant was added to 300 μL of buffer 2 (50 mM HEPES, 30 mM KPI, 0.4 mM CoA (Sigma Aldrich, #C4282), 3 mM NAD+ (Sigma Aldrich, #N0632), 5% FBS, 2 mM Thiamine (Sigma Aldrich, #T1270), 2 mM magnesium chloride and 7.8 μM [14C] valine (Perkin Elmer, #NEC291EU050UC, Waltham, MA)). This reaction took place in a 1.5‐mL Eppendorf tube containing a raised 2‐M NaOH wick trap. Each Eppendorf tube was sealed tight and placed in a shaking incubator at 37°C for 30 min. The radiolabeled 14CO2 contained in the wick trap was counted in a liquid scintillation counter.
L2884
Manufactured by Merck Group
L2884 is a laboratory equipment product. It is designed for general laboratory use. The core function of this product is to provide a controlled environment for various scientific experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Leupeptin, by Merck Group (3 mentions)
A1153, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
St506, by Beyotime (2 mentions)
N0632, by Merck Group (1 mentions)
Nec291eu050uc, by PerkinElmer (1 mentions)
Bio gen pro200 homogenizer, by PRO Scientific (1 mentions)
Phenanthroline, by Merck Group (1 mentions)
P7626, by Merck Group (1 mentions)
D0632, by Merck Group (1 mentions)
P5318, by Merck Group (1 mentions)
E9884, by Merck Group (1 mentions)
D6750, by Merck Group (1 mentions)
Anti α tubulin dm1α, by Merck Group (1 mentions)
Protran ba83, by Cytiva (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Picrotoxin, by Merck Group (1 mentions)
Roscovitine, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
7 protocols using l2884
1
Measuring BCKD Activity in Muscle and Liver
2
Ex Vivo Autophagy Flux Assay
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The following protocol was adapted from previous studies performing ex vivo autophagy flux in rodents [21 (link),36 (link)]. Upon collection of the skeletal muscle sample, two small pieces (~20 mg all together) were placed in 3 mL of oxygenated DMEM CO2 independent media (ThermoFisher #18045088) at 37 °C. The tissues were then incubated with continuous oxygenation for 1 h with (‘treated’ sample, with inhibitors) or without (‘untreated’ sample) 60 µL of NH4Cl (20 µL·mL−1; 40 mM; Merck #101142) and 30 µL Leupeptin (10 µL·mL−1; 100 uM; Sigma Aldrich #L2884). Upon completion of a 1-h incubation, samples were snap-frozen and stored at −80 °C until further analysis. Autophagy flux (Net LC3B-II flux) was obtained by subtraction of the densitometric value of LC3B-II from treated compared to the untreated sample.
3
Drosophila Protein Extraction and Immunoblotting
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For each genotype, 15 brains from third-instar larvae were dissected in PBS, 1% protease inhibitor cocktail and extracted in 100 μl of cold protein extract buffer (50 mM trisHCl pH 7.4 (200923A, Euromedex), 50 mM NaCl (27810, Prolabo), 1 mM EDTA (E9884, Sigma), 0.25% DOC (D6750, Sigma), 0.1% SDS (EU0660, Euromedex), 1 mM PMSF (P7626, Sigma), 1‰, DTT (D0632), 1% of protease inhibitor solution (1 mM benzamidine-HCl (B6506, Sigma), 0,1 mg ml−1 phenanthroline (131377, Sigma), 1 mg ml−1 aprotinin (A6103, Sigma), 1 mg ml−1 leupeptin (L2884, Sigma), 1 mg ml−1 pepstatin A (P5318, Sigma)). Protein extracts were separated by SDS–polyacrylamide gel electrophoresis with either a 10% BIS-TRIS or a 3-8% TRIS-Glycine NuPAGE gel (Invitrogen) and then transferred on a nitrocellulose membrane (Protran BA83, Whatman). Membranes were probed with antibodies anti-Fzy (1:1,000, a gift from J. Raff lab) and anti-α-tubulin (DM1Α) (1:1,000, Sigma-Aldrich).
4
Autophagic Flux Monitoring in Sepsis
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Autophagic flux was monitored in 14–16 weeks old male wild-type C57BL6/J mice subjected to sham or CLP procedures. Briefly, mice received an i.p. injection of the autophagy inhibitor leupeptin (40 mg/kg dissolved in sterile PBS (Sigma-Aldrich # L2884) 20 h post sham or CLP procedures. The sham and CLP control groups received an equivalent volume of sterile PBS. Mice were euthanized 4 hours post leupeptin injection (24h post sham or CLP surgery). Muscles were then rapidly excised and frozen in liquid nitrogen and used for immunoblotting to detect LC3B proteins. Data are shown in Figures S3 E–S3F.
5
Hippocampal and Cortical Cell Stimulation
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To examine downscaling processes, DIV18 or DIV19 hippocampal cells were stimulated with either picrotoxin (100 μM; Sigma-Aldrich) or equivalent volume (1:500) of ethanol absolute for 48 h.
For investigating CDK5 inhibition, DIV19 hippocampal cells were stimulated with either roscovitine (10 μM, R7772; Sigma-Aldrich) or equivalent volume (1:1,000) of DMSO for 18 h.
For studying SPAR and GluA1 degradation, DIV5 cortical cells were stimulated with either leupeptin (200 μg/ml, L2884; Sigma-Aldrich) or equivalent volume (1:250,000) of water for 20–21 h.
For investigating CDK5 inhibition, DIV19 hippocampal cells were stimulated with either roscovitine (10 μM, R7772; Sigma-Aldrich) or equivalent volume (1:1,000) of DMSO for 18 h.
For studying SPAR and GluA1 degradation, DIV5 cortical cells were stimulated with either leupeptin (200 μg/ml, L2884; Sigma-Aldrich) or equivalent volume (1:250,000) of water for 20–21 h.
6
Protein Expression Analysis by Western Blot
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Western blot analyses were performed as previously described (35). Briefly, cells were washed 3 times with PBS and lysed at 4°C in RIPA buffer (P0013C, Beyotime Biotechnology) containing phenylmethylsulfonyl fluoride (0.5 mM, ST506, Beyotime Biotechnology), aprotinin (5 μg/mL, A1153, Sigma-Aldrich), and leupeptin (5 μg/mL, L2884, Sigma-Aldrich). Approximately 50 μg of protein was separated by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies at the following dilutions: p65 (1:1000 dilution; 6956), p38 (1:1000 dilution; 14,451), JNK (1:1000 dilution; 9252), p-p38 (1:1000 dilution; 9215s), p-JNK (1:1000 dilution; 9251), p-ERK1/2 (1:1000 dilution; 4370T) and β-actin (1:1000 dilution; 4970, Cell Signaling Technology). The blots were then incubated for 1 h at room temperature with the appropriate secondary antibodies conjugated to horseradish peroxidase (1:2000 dilution; ZSGB-Bio). Proteins were visualized using an enhanced chemiluminescent detection reagent (6683, Signaling Technology). Densitometric analysis was performed with Image-J software, and target protein expression was normalized to β-actin expression.
7
Western Blotting Analysis of Cellular Proteins
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Western blot analyses were performed as previously described (Lin et al., 2018) (link). Briefly, cells were washed 3 times with PBS and were lysed at 4°C with RIPA buffer (P0013C, Beyotime Biotechnology) containing phenylmethylsulfonyl fluoride (0.5 mM, ST506, Beyotime Biotechnology), aprotinin (5 μg/mL, A1153, Sigma-Aldrich), and leupeptin (5 μg/mL, L2884, Sigma-Aldrich). Approximately 35 μg of proteins was separated on 10% SDS-PAGE and blotted onto nitrocellulose membrane. Membranes were blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 and incubated overnight at 4°C with primary antibodies at the following dilutions: CIDEC (1:1,000 dilution; ab198204, Abcam), FASN (1:1,000 dilution; Cell Signaling Technology), C/EBPβ (1:1,000 dilution; ab264305, Abcam), and β-actin (1:1,000 dilution; 4970, Cell Signaling Technology). The blots were then incubated with the appropriated secondary antibodies conjugated to horseradish peroxidase (1:500 dilution; ZSGB-Bio) at room temperature for 1 h. Proteins were visualized using enhanced chemiluminescent detection reagent (6683, Signaling Technology). Densitometric analysis was performed with Image-Pro Plus 6.0 (Media Cybernetics Inc.), and target protein expression was normalized to β-actin expression.
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