Total cell lysates were prepared from seven OD600 cultures by incubating on ice for 1 h in 10% trichloroacetic acid. After two acetone wash-sonication cycles, samples were bead-beated for 5 min in 70 µl 2× urea buffer (150 mM Tris, pH 6.8, 6 M urea, and 6% SDS) and incubated 5 min at 37°C. After addition of 70 µl 2× sample buffer (150 mM Tris, pH 6.8, 2% SDS, 100 mM DTT, and bromophenol blue), samples were bead-beated for 5 min and heated for 5 min at 37°C. Samples were run on 10% polyacrylamide gels and transferred to nitrocellulose membranes. Antibodies used for blotting were G6PDH (A9521; Sigma-Aldrich), GFP (B2; Santa Cruz Biotechnology, Inc.), and Vph1 (10D7, Invitrogen). Antibodies against Dsc2, Dsc3, Ubx3, and Tul1 were provided by P. Espenshade (Johns Hopkins University, Baltimore, MD).
G6pdh
Manufactured by Merck Group
Sourced in United States
G6PDH is a laboratory equipment product developed by Merck Group. It is an enzyme that catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-delta-lactone, which is the first step in the pentose phosphate pathway. This product is used in various biochemical and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
H3k36me3, by Abcam (4 mentions)
Flag tag (flag), by Merck Group (4 mentions)
Glucose 6 phosphate (g6p), by Merck Group (4 mentions)
Gfp clone jl 8, by Takara Bio (2 mentions)
Leuconostoc mesenteroides, by Merck Group (2 mentions)
H3k36me2, by Active Motif (2 mentions)
Hexokinase, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Hydrazine, by Merck Group (2 mentions)
H2b k123ubq1, by Cell Signaling Technology (2 mentions)
H3k4me3, by EpiCypher (2 mentions)
H3k79me3, by Abcam (2 mentions)
H3k79me2, by Active Motif (2 mentions)
Ecl prime, by Cytiva (2 mentions)
Gfp b 2, by Santa Cruz Biotechnology (1 mentions)
Prism 5, by GraphPad (1 mentions)
Clb2 y 180, by Santa Cruz Biotechnology (1 mentions)
Mini beadbeater, by Biospec (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
31 protocols using g6pdh
1
Protein Extraction and Western Blotting
2
Assessing In Vitro Efficacy of Compounds
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The in vitro efficacy of twelve compounds was assessed in two separate steps: a SC50 assay to evaluate the potency of each compound at a unified compound concentration of 50 μM. Then, we seek to obtain the values of EC50 and kinetic parameters Ka, Vmax of promising compounds, according to a reported method with minor modifications10 (link). Briefly, Twelve compounds unified in the same concentration of 50 μM were assayed in a 96-well plate with a final reaction volume of 100 μl. The reaction mixture consisted of 25 mM Hepes (pH 7.5), 10 mM glucose, 1 mM ATP, 4 U/ml G6PDH (Sigma-Aldrich Co. LLC., St. Louis, MO, USA), 1 mM NADP, 2.5 mM MgCl, 50 mM KCl, 2 mM DTT, 1 mM recombinant human glucokinase and test compounds. The whole reaction mixtures were kept under 37 °C for 20 minutes. The increase in the rate of absorbance of NADPH generated in the reactions was monitored kinetically at 340 nm. The values were calculated by comparing with untreated GK. This term is called the efficacy of stimulatory concentration (SC50). Then, the EC50 of promising candidates were determined in the presence of various fixed concentrations of compound 7 (2–300 μM). Values were calculated by fitting enzymatic rates to the Hill equation by using GraphPad Prism 5.01.
3
Western Blotting of Protein Samples
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Samples were prepared for Western Blotting by resuspending equivalent optical densities of cells in preheated SDS sample buffer (50 mM Tris pH 7.5, 5 mM EDTA, 5% SDS, 10% glycerol, 0.5% β-mercaptoethanol, bromophenol blue, 1 μg/ml leupeptin, 1 μg/ml bestatin, 1 mM benzamidine, 1 μg/ml pepstatin A, 17 μg/ml PMSF, 5 mM sodium fluoride, 80 mM β-glycerophosphate and 1 mM sodium orthovanadate), followed by incubation at 95°C for 5 minutes. Glass beads were then added and samples were bead beat using a Biospec Mini-Beadbeater for 3 minutes. Samples were clarified by centrifugation and analyzed by SDS-PAGE followed by Western blotting. Western blots were carried out with antibodies against GFP (clone JL-8, Clontech), Clb2 (y-180, Santa Cruz Biotechnology), Cdc28/Cdk1 (yC-20, Santa Cruz Biotechnology), HA (clone 12CA5), V5 (ThermoFisher), Myc (clone 9E10, Covance), and G6PDH (Sigma). Where indicated, quantitation was performed using a BioRad ChemiDoc Touch imaging system and the accompanying ImageLab software.
4
Assaying PGI Activity in Drosophila
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PGI activity in KB1_0553 (DmPMI) was tested using two standard assays (Hansen et al., 2004b ) for the reaction described by the following equations:
Assay 1:F 6 P ⇔ P G I G 6 P G 6 P + N A D P + ⇔ G 6 P D H 6 P G 15 L + N A D P H
Assay 2:G 6 P ⇔ P G I F 6 P F 6 P + N A D H ⇔ M 1 P D H M 1 P + N A D + 1984 ) with > 95% purity (see Fig. S1 ). The reactions were started by adding 1 μg of purified protein into the reaction mixture, and the product formation was inferred from the measurement of NADPH formation in assay 1 and of oxidation of NADH in assay 2 with a spectrophotometer at 340 nm absorbance and 30°C.
Assay 1:
Assay 2:
5
Yeast Cyclin C Oxidative Stress Response
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Yeast strains expressing myc-tagged cyclin C were grown in 0.5-l cultures at 30°C to 0.5 OD600/ml in SCD without leucine and treated with 0.2 mM H2O2 as noted. Cells were pelleted and washed in H2O and then in yeast extract buffer (YEB; 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–KOH, pH 7.1, 100 mM β-glycerolphosphate, 50 mM NaF, 5 mM ethylene glycol tetraacetic acid, 5 mM EDTA, 10% glycerol, 0.25% Tween 20, and 150 mM KCl). Pellets were resuspended 1:1 (wt/vol) in YEB containing protease inhibitors (cocktail tablet; Roche), 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride, and cell lysates were frozen into pellets by dripping into liquid nitrogen. These pellets were then beat in a freezer mill (6970EFM; SPEX Sample Prep) three times for 1 min. On thawing, the lysate was spun two times for 20 min at 14,000 × g at 4°C. Two milligrams of total protein was incubated with α-myc antibody (9E10; Covance) and rotated for 2 h at 4°C before incubation with YEB-washed Protein G Sepharose beads. Bound beads were resuspended in SDS-sample buffer and boiled to remove bound protein. “Input” samples of 50 μg of total yeast protein and “precipitation” samples were separated by 10% SDS–PAGE, followed by Western blotting using the same anti-myc antibody (1:1000) and anti–glucose-6-phosphate dehydrogenase (G6PDH; 1:100,000; Sigma-Aldrich).
6
Reactivating Recombinant Luciferase and G6PDH
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Recombinant luciferase from P. pyralis (Sigma-Aldrich) was aggregated and reactivated by the Hsc70 system as in previous works32 (link), as it was Glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides (Worthington)46 (link). Reactivation percentages were calculated considering the activity of the aggregated and native (in the presence of chaperones) proteins as 0% and 100%, respectively.
7
Antibody Generation and Reagent Sources
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The following antibodies were from Sigma-Aldrich: G6PDH produced in rabbit (A9521), HA-peroxidase (H6533), and FLAG M2 produced in mouse (F1804). The antibody for Por1 produced in mouse (459500) was from Thermo Fisher Scientific. The synthetic peptides RKDIGDDKPK (amino acid residues 731–740 of Yme1), ENETLLDRIVALKD (amino acid residues 54–67 of Tom22), and KEDALSLKDALLGV (amino acids residues 64–77 of Om45) were used for generating Yme1, Tom22, and Om45 antibodies, respectively.
Yeast extract, peptone, and yeast nitrogen base without amino acids were from BD. Yeast complete supplement mixture was from MP Biomedicals. Yeast amino acid dropout supplements were from Takara Bio Inc. CHX was from Amresco. Other chemicals or reagents were from Sigma-Aldrich if not otherwise indicated.
Yeast extract, peptone, and yeast nitrogen base without amino acids were from BD. Yeast complete supplement mixture was from MP Biomedicals. Yeast amino acid dropout supplements were from Takara Bio Inc. CHX was from Amresco. Other chemicals or reagents were from Sigma-Aldrich if not otherwise indicated.
8
Western Blot Antibody Panel for Cell Signaling
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Primary antibodies against GFP (Takara Bio, 632380, AB_10013427; 1:5000), G-6-PDH (Sigma-Aldrich, A9521, AB_258454; 1:5000), Adh1 (Millipore, 126745, AB_564196; 1:200000), RPS6 (Cell Signaling Technology, 2217, AB_331355; 1:1000), phospho-RPS6 (Ser235-236) (Cell Signaling Technology, 4856, AB_2181037; 1:1000), RPS6KB (Cell Signaling Technology, 2708, AB_390722; 1:1000), phospho-RPS6KB (Thr389) (Cell Signaling Technology, 9205, AB_330944; 1:1000), EIF4EBP1 (Cell Signaling Technology, 9452, AB_331692; 1:1000), phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855, AB_560835; 1:1000), phospho-EIF4EBP1 (Ser65) (Cell Signaling Technology, 9451, AB_330947; 1:1000), AKT1 (Cell Signaling Technology, 4691, AB_915783; 1:1000), phospho-AKT(Ser473) (Cell Signaling Technology, 4060, AB_2315049; 1:1000), phospho-AKT(Thr308) (Cell Signaling Technology, 13038, AB_2629447; 1:1000), ULK1 (Cell Signaling Technology, 8359, AB_11178668; 1:1000), phospho-ULK1(Ser757) (Cell Signaling Technology, 6888, AB_10829226; 1:1000), MAP1LC3 AB (Cell Signaling Technology, 12741, AB_2617131; 1:1000), β-actin (Cell Signaling Technology, 4967, AB_330288; 1:1000) were used.
9
Western Blot Analysis of Histone Methylation
10
Enzymatic Assays for Phosphorylation Reactions
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PMI activity in KB1_0553 (DmPMI) was tested with two standard assays (Hansen et al., 2004a ) for the reactions described by the following equations:
Assay 1:M 6 P ⇔ P M I F 6 P F 6 P + N A D H ⇔ M 1 P D H M 1 P + N A D +
Assay 2:M 6 P ⇔ P M I F 6 P F 6 P ⇔ P G I G 6 P G 6 P + N A D P + ⇔ G 6 P D H 6 P G 15 L + N A D P H 1984 ) with > 95% purity (see Fig. S1 ). In assay 2, the reaction mixture contained 100 mM Tris‐HCl (pH 7.5), 0.5 mM NADP+, 10 mM M6P and 1.1 U of G6PDH (Sigma‐Aldrich, St. Louis, MO) and 1 U of PGI (Sigma‐Aldrich, St. Louis, MO). The reactions were started by adding 1 μg of purified protein into the reaction mixture, and the product, F6P formation, was measured by coupling it to the oxidation of NADH in assay 1 and to the formation of NADPH in assay 2. In both instances, the product formation was inferred from the measurement of absorbance with a spectrophotometer at 340 nm and 30°C.
Assay 1:
Assay 2:
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