Murine thoracic arteries were ground in liquid nitrogen and the resulting powder was lysed in RIPA-buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) Igepal CA-630, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) sodium dodecyl sulfate) for 30 min on ice. After removing cellular debris by centrifugation (16.000× g, 15 min, 4 °C), protein concentrations were measured using the Bradford reagent (BioRad, Munich, Germany). Endothelial cells were washed with PBS and scraped off the dishes on ice. After centrifugation, the resulting cell pellet was lysed in RIPA-buffer for 30 min on ice. After removing cellular debris by centrifugation (16.000× g, 15 min, 4 °C), protein concentrations were measured using the Bradford reagent (BioRad, Munich, Germany). Immunoblotting was performed with antibodies directed against eNOS (1:500) (Abcam, Cambridge, UK), phospho-eNOS (S1177) (1:500), phospho-eNOS (T495) (1:500) and the myc-tag (1:500) (all Cell Signaling Technology, Frankfurt, Germany). Blotting membranes were incubated with primary antibodies overnight at 4 °C before they were washed and incubated with secondary antibodies according to standard procedures. Detection was performed by enhanced chemiluminescence using the ECL reagent (GE Healthcare, Freiburg, Germany) and standard X-ray films. Semi-quantitative analyses were performed on scanned X-ray films using ImageJ 1.42q60 .
Bradford reagent
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The Bradford reagent is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Pvdf membrane, by Merck Group (42 mentions)
Bovine serum albumin (bsa), by Merck Group (42 mentions)
Protease inhibitor cocktail, by Merck Group (39 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Protease inhibitor cocktail, by Roche (25 mentions)
Ripa buffer, by Merck Group (23 mentions)
β actin, by Cell Signaling Technology (23 mentions)
Pvdf membrane, by Bio-Rad (22 mentions)
Nitrocellulose membrane, by Bio-Rad (22 mentions)
β actin, by Santa Cruz Biotechnology (18 mentions)
Protease inhibitor, by Merck Group (16 mentions)
β actin, by Merck Group (15 mentions)
Western blotting plus chemiluminescence reagent, by Merck Group (15 mentions)
Protease inhibitor, by Roche (14 mentions)
Ezripa lysis kit, by ATTO Corporation (14 mentions)
Trans blot turbo transfer system, by Bio-Rad (13 mentions)
Nitrocellulose membrane, by Cytiva (13 mentions)
Hybond, by Cytiva (12 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (12 mentions)
Clarity western ecl substrate, by Bio-Rad (12 mentions)
1 210 protocols using bradford reagent
1
Murine Thoracic Artery Protein Analysis
2
Prostate Cancer and Pheochromocytoma Cell Line Cultures
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LNCaP cells, a human prostate cancer cell line, and PC12 cells, a pheochromocytoma cell line derived from rat adrenal medulla were both obtained from American Tissue Culture Collection (ATCC, Washington DC; LNCaP, CRL-1740 and PC-12, CRL-1721). Cell culture media including fetal bovine serum (FBS), and other supplements were from Invitrogen (Grand Island, NY). Antibodies for Western Blot and markers were obtained from Santa Cruz Biotechnology and DMSO, protein standards and 1X Bradford reagent were from Bio-Rad (Hercules, CA). See Fig. 1 .Fig. 1 ![]()
PC-12 and LNCaP cells in culture. (A) PC-12 cells, at 1000×, in 10% FBS-DMEM, supplemented with NGF. (B) LNCaP, at 200×, cells grown in 10% FBS-DMEM.
3
Cloning and Purification of Recombinant EspR
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The EspR ORF was amplified from Mtb H37Rv genomic DNA and cloned in pCDNA3.1 using BamH1 and XhoI sites (pC-EspR). pET-EspR was generated by cloning EspR ORF in pET28a using BamH1 and XhoI. pMSP12::mCherry-EspR was generated by cloning EspR ORF in pMSP12::mCherry (procured from the Addgene #30169) using KpnI and HindIII sites. pET-EspR transformed E. coli BL-21λDE3 (Agilent technologies, USA) cells were used for purification of recombinant MtbEspR protein (carrying N and C terminal 6X histidine tag) upon induction with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fermentas, USA) for 4 h at 37°C under native conditions using Talon-affinity resin (Clontech, USA) according to the manufacturer’s protocol and eluted with 200 mM imidazole containing PBS. Subsequently, rMtbEspR was dialyzed against PBS supplemented with 50 mM NaCl, and 10% glycerol. The protein concentration was measured using 1X Bradford Reagent (Bio-Rad, USA) according to the manufacturer’s instructions and the integrity of the protein was checked on 15% SDS PAGE (Supplementary Figures S1A, B
4
Protein Extraction and Proteomics Analysis
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The protein concentration was determined after protein extraction using the Bio-Rad Bradford reagent (Bio-Rad, Munich, Germany) and bovine serum albumin as protein standard. Protein samples of solubilized membrane extract and soluble fractions were subjected to SDS-PAGE. Per lane, 50 μg of protein lysates was applied. Afterwards, each sample lane was cut into four slices and prepared for proteolytic cleavage using trypsin (Promega, Madison, WI, USA)48 (link). Peptide lysates were extracted and desalted using C18 ZipTips (Merck Millipore, Darmstadt, Germany).
5
Serine Acetyltransferase Enzymatic Activity Assay
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Serine acetyltransferase enzymatic activity assays were performed as described previously (Watanabe et al., 2008b (link)). Frozen ground material (20 mg) was homogenized in 100 μL of extraction buffer containing 250 mM potassium phosphate, pH 8.0, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol. The soluble protein amount was measured with the Bio-Rad Bradford reagent (Bio-Rad Laboratories) according to the manufacturer’s instructions. The enzymatic activity of SERAT was determined in reaction mixtures (100 μL) containing 50 mM Tris–HCl, pH 8.0, 1 mM acetyl-CoA, 10 mM serine. The reaction was performed at 30°C for 15 min and terminated by the addition of 10 μL of 7.5% (w/v) trichloroacetic acid. SERAT activity was determined for the production of OAS, which was derivatized with O-phthalaldehyde using HPLC (see section “Determination of Amino Acid Contents”).
6
Apoptosis Pathway Regulation Assay
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All chemicals and reagents used were of analytical grade and were purchased from Sigma Aldrich (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit and 7-aminoactinomycin D (7-AAD) were received from Biolegend (San Diego, CA, USA). Antibodies for immunoblotting including PARP (Santa Cruz, sc-25780), cPARP (Cell Signaling, D64E10), laminA/C (Abcam, ab133269), caspase-8 (MBL, M032-3), caspase-10 (MBL, M059-3), caspase-9 (MBL, M054-3), caspase-3 (Cell signaling, 9665), anti-DR4 (Abcam, ab8415), anti-DR5 (Abcam, ab8416), GAPDH (Santa Cruz, sc-47724), HSC70 (Santa Cruz, sc-7298), BiP-1 (Cell signaling, C50B12), IRE1 (Cell signaling, 14C10), RIPK1 (BD Bioscience, 610459), XIAP (Santa Cruz, sc-55550), and survivin (Abcam, EP2880Y). Bradford reagent was obtained from Bio-Rad (Marnes-la-Coquette, France). Chemiluminescent Western blots detection kit was purchased from Advansta (San Jose, CA, USA).
7
Had1-FLAG Mobility Assay Protocol
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Had1 mobility assay was conducted as described previously (Park et al. 2016 (link)). Strains expressing Had1-FLAG were grown in YPD at 25° to an optical density OD600 0.6–0.8, and cultures were grown at 25° or shifted from 25 to 37° for 1 hr with or without FK506 (2 μg/ml). Cells were collected and disrupted in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100) supplemented with a protease inhibitor tablet (Roche) and phosphatase inhibitor cocktails (Thermo) using a bead beater for 10 cycles (60 sec homogenization with 60 sec rest). Cell lysates were centrifuged for 15 min at 14,000 × g, the supernatant was recovered, and protein concentration was determined by employing the Bio-Rad Bradford reagent. The supernatant was subjected to SDS-PAGE and transferred to PVDF membranes (Bio-Rad). For western blot analysis, we employed mouse monoclonal anti-FLAG M2 antibodies (Sigma), anti-mouse antibody conjugated to horseradish peroxidase (Thermo), and ECL western blotting detection reagent (GE healthcare).
8
Liver Protein Extraction Protocol
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Protein for three of the individuals included in the study was not available for the RYBG time point. Protein was extracted by pulverizing 3-4 mg of frozen liver under liquid nitrogen. The resultant powder was incubated with buffer containing Complete Protease Inhibitor Cocktail (Roche), 30 mM TrisCl, 7 M urea, 2 M thiourea and 4% (w/v) CHAPS) for 45 minutes on ice, with vigorous vortexing every 15 minutes. The mixture was centrifuged at 14000 g for 10 minutes to remove any insoluble material. The supernatant was aliquoted for storage at −80°C. Protein was quantified using Bradford reagent (Bio Rad) on a Bio-Rad Benchmark microplate reader [27 (link)].
9
Protein Extraction and Western Blot
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Western blot was performed as described.16 Cultured cells or tissues were first lysed in RIPA buffer for 15 minutes on ice. Cell lysates were clarified after centrifugation at 20 800 g for 15 minutes. Protein concentration was determined using Bradford Reagent (Bio‐Rad). Equal amounts of protein lysates were resolved by SDS‐PAGE. After transferring membranes, the samples were immunoblotted with indicated primary and corresponding secondary antibodies.
10
Stoichiometry Quantification of Acetylation
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