All Caulobacter strains were grown overnight in liquid PYE media. After overnight growth, cells were back diluted to OD600 0.1 and outgrown to mid-exponential phase before being normalized to OD600 0.1 and 10-fold serially diluted on to media. For experiments using mitomycin C (Sigma, St. Louis, MO) and L-canavanine (Sigma), drugs were prepared at a stock concentration of 400 μg/mL mitomycin C, and 100 mg/ml L-canavanine and filter sterilized. PYE agar was cooled before the drugs were added and plates were left to air dry prior to serial dilution plating. All plates were incubated at 30°C for 2–3 days and imaged with a Syngene G:Box.
L canavanine
Manufactured by Merck Group
Sourced in Germany, United States
L-canavanine is a laboratory reagent that serves as a structural analog of the amino acid L-arginine. It is commonly used in biochemical and cell biology research to study the effects of arginine analogues on various cellular processes and pathways.
Automatically generated - may contain errors
Lab products found in correlation
Sulforhodamine b, by Merck Group (2 mentions)
Caffeine, by Merck Group (2 mentions)
Sucrose, by Merck Group (2 mentions)
Uracil, by Merck Group (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Csm ura, by Formedium (2 mentions)
Csm arg, by Formedium (2 mentions)
Yeast nitrogen base, by Formedium (2 mentions)
Canavanine, by Merck Group (2 mentions)
5 fluoroorotic acid, by US Biological (2 mentions)
Azathioprine, by Merck Group (1 mentions)
Esculin hydrate, by Merck Group (1 mentions)
Cinnamaldehyde, by Merck Group (1 mentions)
P benzoquinone, by Merck Group (1 mentions)
Catechin hydrate, by Merck Group (1 mentions)
Curcumin, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Denatonium, by Merck Group (1 mentions)
Tricholine citrate, by Merck Group (1 mentions)
28 protocols using l canavanine
1
Caulobacter Growth and Drug Sensitivity
2
Compound Preparation for Biological Assays
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Curcumin, azathioprine, resveratrol, catechin hydrate, baicalin hydrate, L-canavanine, 4-nitropyridine N-oxide, p-benzoquinone, esculin hydrate, cinnamaldehyde, and ciprofloxacin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Stock solutions of ciprofloxacin and the compounds were prepared in dimethyl sulfoxide (pure DMSO), sterile water, or hydrochloric acid according to the manufacturer’s recommendations.
3
Characterization of Diverse Compounds
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Sucrose, denatonium, quinine, papaverine, caffeine, strychnine, L -canavanine, N,N-diethyl-m-toluamide (DEET), sulforhodamine B, KCl and tricholine citrate were purchased from Sigma-Aldrich (Saint Louis, MO). Berberine sulfate trihydrate and Brilliant Blue FCF were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
4
Inhibition of iNOS Modulates Cell Cycle and Apoptosis in HCC
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The primary antibody against DNM3 (cat. no. ab3458) was obtained from Abcam (Cambridge, UK). Primary antibodies against inducible (i)NOS (cat. no. sc-7271), nNOS (cat. no. sc-136006) and β-actin (cat. no. sc-130300) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primary antibodies against caspase-3 (cat. no. 9662), cleaved caspase-3 (cat no. 9664), caspase-9 (cat. no. 9508), cleaved caspase-9 (cat. no. 7237), poly ADP-ribose polymerase (PARP; cat. no. 9532), cleaved PARP (cat. no. 5625), cyclin D1 (cat. no. 2978), cyclin-dependent kinase (CDK)2 (cat. no. 2546), CDK4 (cat. no. 12,790) and GAPDH (cat. no. 5174) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The iNOS inhibitor L-canavanine was supplied by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). L-canavanine (1 mM) was incubated with the indicated cells at 37°C for 24 h. The L02 human hepatic cell line and HepG2, Hep3B, SMMC-7721, PLC/PRF/5, Bel-7402 and Huh7 human HCC cell lines were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum and maintained in 5% CO2/95% air at 37°C.
5
Reversion Frequency and Canavanine Resistance
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For reversion frequency of Trp-phenotype and the occurrence of Canavanine resistant clones three independent cultures of the considered strains were cultivated at 28 °C in SD medium and auxotrophic requirement as needed. For each culture, three plates of selective medium lacking tryptophan or containing L-canavanine (3 µg/ml Sigma-Aldrich S.r.l. Milan, Italy) were inoculated and incubated at 28 °C for 3–5 days. The frequency of the Trp + reversion and of Canavanine resistant mutants was normalized to the number of viable cells in each experiment. Viability at day 1 was determined with the microcolonies test [25 (link)], while for the following days a standard colony forming units on YPD plates was employed. To measure RNA oxidation and Trp + reversion rate in the presence of antioxidant compounds 1 mM ALC and 26 mg/mL apple extracts [21 (link)] were added to the Kllsm4Δ1cell cultures. Total RNAs were extracted after 1 day of growth and analysed by Northwestern as described. Trp + reversion frequency was determined in the absence or in the presence of antioxidants.
6
Purification of Recombinant Proteins
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l -Canavanine, tris(2-carboxyethyl)phosphine
(TCEP),l -Arg, sodium phosphate monobasic, and imidazole
were acquired from Sigma (St. Louis, Missouri). Kanamycin, isopropyl
β-d -1-thiogalactopyranoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), and Ni-NTA agarose beads were purchased from GoldBio
(St. Louis, Missouri). 2OG was purchased from Fluka. EDTA was purchased
from Invitrogen. 50% PEG 3350, PEG 400, PEG MME 550, and 50% ethylene
glycol were purchased from Rigaku or Hampton. All other chemicals
were reagent grade or better.
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(TCEP),
were acquired from Sigma (St. Louis, Missouri). Kanamycin, isopropyl
β-
acid (HEPES), and Ni-NTA agarose beads were purchased from GoldBio
(St. Louis, Missouri). 2OG was purchased from Fluka. EDTA was purchased
from Invitrogen. 50% PEG 3350, PEG 400, PEG MME 550, and 50% ethylene
glycol were purchased from Rigaku or Hampton. All other chemicals
were reagent grade or better.
7
Yeast Culture Media and Selection Conditions
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Media used in this study consisted of 1% yeast extract, 2% peptone, and 2% glucose (YPD). Plates of these media were made with 1.5% agar for standard growth conditions. YPD containing Hygromycin B (Invitrogen) (200 mg/L), G418 (Formedium) (200 mg/L), or ClonNat (100 mg/L) were used for selection of yeast transformants. Synthetic complete media consisted of 6.7 g/L Yeast Nitrogen Base with ammonium sulfate and without amino acids, 1.77 g/L CSM-Ura (Formedium), 50 mg/L uracil (Sigma), and 2% glucose. Canavanine plates consisted of 6.7 g/L Yeast Nitrogen Base with ammonium sulfate and without amino acids, 0.74 g/L CSM-Arg (Formedium), 60 mg/liter l -canavanine (Sigma-Aldrich), 1.5% agar, and 2% glucose. FOA plates consisted of 0.67% Yeast Nitrogen Base with ammonium sulfate and without amino acids, 0.2% CSM-Ura (Formedium), 50 mg/L uracil (Sigma-Aldrich), 1 g/L FOA (Formedium), 1.5% agar, and 2% glucose. Ethanol and methanol were purchased from VWR in their highest purity; isopropanol was purchased from Sigma-Aldrich.
8
Quantification of Yeast Mutation Frequency
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Yeast colonies were picked, suspended in 3 mL SC medium with 2% glucose and without leucine and uracil, and grown to a stationary phase. The cells were then pelleted, washed twice in sterile water, and then resuspended in SC induction medium with 2% galactose and 1% raffinose, but without leucine and uracil, to an OD600 of 0.3. The cells were incubated for 20 h prior to plating on YPAD rich or SC media plates without arginine but with 60 mg/mL l -canavanine (Sigma). After incubation for 3 days, the colony number on each plate was counted. The C-to-T mutation frequency in CAN1 was determined as the ratio of the colony count on canavanine-containing plates to the colony count on YPAD-rich media plates. Each experiment was performed at least three times on different days. To determine the mutation spectrum, colonies were randomly picked and suspended in sterile water, followed by PCR amplification of the relevant CAN1 fragment and DNA sequencing. Control cultures (not treated with base editors) did not produce canavanine-resistant colonies.
9
Measuring Mutation Rates in Yeast
10
Yeast Genetic Manipulation Techniques
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Yeast strains are described in Supplementary Table 4 . Plasmid cloning work and circuit construct characterization were all performed in Escherichia coli DH10B strains, which were cultured in LB (Luria–Bertani broth) media (10 g l−1 peptone, 5 g l−1 NaCl, 5 g l−1 yeast extract). S. cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used as the initial host for most DNA assembly and transformation in this study. Yeast strains were cultured in YPD medium (10 g l−1 yeast extract, 20 g l−1 peptone, and 20 g l−1 glucose), and SC-His (synthetic complete medium lacking histidine with 20 g l−1 glucose) and (synthetic media lacking histidine with 20 g l−1 galactose); β-Estradiol and l -canavanine were purchased from Sigma-Aldrich. SC-Canavanine plate (synthetic complete medium lacking arginine with 20 g l−1 glucose and 60 μg ml−1l -canavanine) were used to select for pathway integration CAN1 locus. Presporulation medium (50 g l−1 glucose, 30 g l−1 Difco nutrient broth and 10 g l−1 Difco yeast extract), sporulation media (10 g l−1 potassium acetate, 0.05 g l−1 zinc acetate dehydrate, add uracil, histidine, leucine at 1 × concentrations), and 1 × stock solution of zymolyase (50 µg ml−1 in 1 M sorbitol) were prepared for sporulation and tetrad dissection.
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