Immunoblot analysis was performed using whole cell lysates from HEK-293T cells. Cells were plated in 6-well plates at 80% confluency, transiently transfected with WT CYP17A1 or appropriate variant constructs using the FuGENE HD transfection reagent (Promega) for 48 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0, which was supplemented immediately prior to use with mini protease inhibitor tablet (Roche) and 5 mM N-ethylmaleimide (Sigma). Lysates were sonicated and centrifuged for 20 minutes at 14,000 × g. Supernatants were collected, and protein concentrations were determined using the Bradford protein assay (BioRad, Hercules, CA). Absorbance was measured using PolarStar Omega plate reader. Protein (30–50 μg per lane) was loaded, resolved on a 10% SDS-polyacrylamide gel (ThermoScientific), and transferred onto a PVDF membrane. Blots were probed with a monoclonal anti-CYP17A1 antibody (Origene) and an HRP-conjugated mouse secondary antibody (Cell Signaling). Immunoreactive bands were visualized with the use of enhanced chemiluminescence reagent (Super Signal, Pierce) on film. Transfected cells were treated with MG132 (10 uM) or cycloheximide (25 μM) for the specified amount of time where indicated.
Mini protease inhibitor tablet
Manufactured by Roche
Sourced in France, United States
The Mini Protease Inhibitor Tablets are a laboratory product designed to inhibit proteases, which are enzymes that break down proteins. The tablets provide a convenient and consistent way to inhibit protease activity in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Rnasin, by Promega (2 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Murine rnase inhibitor, by Roche (1 mentions)
Recombinant rnasin ribonuclease inhibitor, by Promega (1 mentions)
Phosstop tablet, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Np 40, by Roche (1 mentions)
Glass bead, by Merck Group (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Ab652, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
15 protocols using mini protease inhibitor tablet
1
Immunoblot Analysis of CYP17A1 Variants
2
Polysome Profiling of Lymphoblastoid Cells
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Six weeks-old LCLs with a density of ∼7.5 × 105 cells/ml were split 1:3 forty eight hours prior to polysome profiling. On the day of the profiling experiment, LCLs were treated with 100μg/ml cycloheximide for 5 min to stall ribosomes on RNA. Cells were pelleted at 4°C and lysed in 250 μl ice-cold polysome lysis buffer (15 mM Tris–HCl pH 7.4; 15 mM MgCl2; 300 mM NaCl; 1% Triton X-100; 0.1% β-mercaptoethanol; 200 U/ml RNasin (Promega); 1 complete Mini Protease Inhibitor Tablet (Roche)/10 ml lysis buffer). Following 10 min incubation at 4°C, the lysate was cleared by centrifugation (10 000 rpm; 4°C; 10 min) and the supernatant was loaded onto a linear sucrose gradient ranging from 17.5–50% (w/v) sucrose (in 15 mM Tris–HCl pH 7.4; 15 mM MgCl2; 300 mM NaCl). Ultracentrifugation was carried out at 4°C at 35 000 rpm for 2.5 h in a SW60Ti rotor. Gradients were fractioned using a Teledyne Isco Foxy Jr. Gradient fractionator, which eluted the gradient into 12 fractions of 400μl volume. In parallel, the polysome profiles were recorded by measuring absorbance at 254 nm. RNA was purified from the fractions using organic solvent extraction followed by isopropanol precipitation.
3
Generation of G3BP1-mEmerald HeLa Cell Lysates
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The generation of cell lysates from G3BP1-mEmerald HeLa cells and the subsequent generation of lysate granules have been described previously15 (link). Briefly, cells were grown to 100% confluency in a 10 cm dish and harvested in 5 mL PBS. All steps were performed at room temperature. Collected cells were spun at 500 × g for 5 min with the supernatant aspirated and discarded. Cell pellets were stored at −80 °C until ready to use. To prepare cell lysates, pellets were thawed for 2 min and resuspended in 250 μL lysis buffer containing 50 mM TRIS pH7.0 (Sigma), 0.5% NP40 (Thermo), 0.025× mini protease inhibitor tablet (Roche) and 40× murine RNase inhibitor (NEB). After a 3-min incubation period lysates were spun at 24,000 × g for 5 min to remove nuclei and cell debris. The supernatant was retained to produce reconstituted stress granules when mixed with unlabelled G3BP1.
4
Polysome Profiling of LPS-Stimulated Macrophages
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RAW264.7 macrophages or BMDM were stimulated with 100 ng/ml LPS (E. coli O111:B4, Sigma L2630) for 1 h. Ribosomes were stalled by addition of 100 µg/ml cycloheximide (CHX) for 5 min, and cells were lysed in Polysome lysis buffer (15 mM Tris-HCl pH 7.4, 15 mM MgCl2, 300 mM NaCl, 1% Triton-X-100, 0.1% β-mercaptoethanol, 200 U/ml RNAsin (Promega), 1 complete Mini Protease Inhibitor Tablet (Roche) per 10 ml). Nuclei were removed by centrifugation (9300× g, 4°C, 10 min) and the cytoplasmic lysate was loaded onto a sucrose density gradient (17.5–50% in 15 mM Tris-HCl pH 7.4, 15 mM MgCl2, 300 mM NaCl and, for fractionation from BMDM, 200 U/ml Recombinant RNAsin Ribonuclease Inhibitor, Promega). After ultracentrifugation (2.5 h, 35 000 rpm at 4°C in a SW60Ti rotor), gradients were eluted with a Teledyne Isco Foxy Jr. system into 16 fractions of similar volume. A rabbit HBB2 in vitro transcript was added to each fraction as a spike-in control and RNA was purified by phenol chloroform extraction. To assess RNA quality and equal purification efficiency across all fractions, the HBB2 in vitro transcript and endogenous Ncl mRNA were detected by Northern blotting.
5
Cardiac Tissue Phosphorylation Analysis
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For analysis of MLP phosphorylation levels, 20 mg of frozen cardiac tissue samples were homogenized into 200 μl freshly prepared ice-cold TLB lysis buffer (20 mM Tris HCL pH 7.6, 138 mM NaCl, 5% glycerol, 1% Triton, 5 mM DTT, 0.5 mM Sodium ortho-vanadate, Mini Protease inhibitor tablet (no EDTA; Roche), Phos-stop tablet (Roche)). Samples were briefly sonicated, incubated on ice for 20 min, centrifuged at 4 °C at 14,000 r.p.m. for 15 min, and supernatants were used for further analysis via Phostag gel analysis58 (link) after determination of protein concentrations. Protein samples with normalized concentrations were supplemented with SDS sample buffer, boiled for 5 min, and either run on a 12% poly-acrylamide SDS-PAGE, or a 12% poly-acrylamide SDS PAGE supplemented with 50 μM Manganese Phostag (WAKO Chemicals). After end of electrophoresis, gels were washed once in immunoblot transfer buffer supplemented with 10 mM EDTA for 10 min, followed by a wash in immunoblot transfer buffer for another 10 min, and prepared for immunoblot transfer and subsequent detection and analysis following standard procedures.
6
Extraction and Analysis of Prion Proteins
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Frozen tissues of adult mice and goats were homogenized in 50 nm Tris HCl, 150 mm NaCl, 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulphate (w/v), 1% of a nonionic nondenaturing detergent (NP‐40), one complete EDTA free mini‐protease inhibitor tablet per 10 mL (Roche Diagnostic, Saint‐Egrève, France). Whole extracts (20 μg of total protein) were subject to 4–15% gradient SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane (GE healthcare Life Sciences, Vélizy‐Villacoublay, France). The membrane was probed with anti‐biotinylated‐PrP antibody (bSha 31; Table 3 ). The secondary antibody used was horseradish peroxydase/streptavidin conjugated antirabbit (Table 3 ). Immunodetection using the enhanced chemiluminescence (ECL) method (PIERCE) was performed according to the manufacturer's instruction and the images were recorded on an image analysis station (Luminescent Image analyse Las‐1000plus Fujifilm).
7
Extraction and Characterization of Bacterial Surface Proteomes
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NCFM in late-log phase (OD 600 0.5˗1.6, Supporting Figure S1) was harvested by centrifugation (3200 x g, 10 min) and washed with 0.9% NaCl. Extracts were prepared by mechanical grinding (5 x 1-min vortex at maximum speed) with a small amount of acid washed glass beads (<100 μm diameter; Sigma) in sample buffer (28 mM Tris-HCl, 22 mM
Tris-base pH 8.5, 100 mM DTT) containing protease inhibitors (complete, MiniProtease
Inhibitor Tablets, Roche). Following heating (100 o C, 2 min) and addition of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT), mixtures were vortexed, centrifuged (10000 x g, 10 min), and supernatants were collected. Surface proteomes were prepared by incubating cell pellets with 5 M lithium chloride (Sigma-Aldrich) (30 min, R.T.)
and collecting supernatants after centrifugation (15000 x g, 15 min) [21] . Proteins were This article is protected by copyright. All rights reserved. 6
precipitated by TCA/acetone [22] , washed with acetone, dissolved in rehydration buffer and concentrations were determined by using 2-D Quant Kit (GE Life Sciences).
Tris-base pH 8.5, 100 mM DTT) containing protease inhibitors (complete, MiniProtease
Inhibitor Tablets, Roche). Following heating (100 o C, 2 min) and addition of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT), mixtures were vortexed, centrifuged (10000 x g, 10 min), and supernatants were collected. Surface proteomes were prepared by incubating cell pellets with 5 M lithium chloride (Sigma-Aldrich) (30 min, R.T.)
and collecting supernatants after centrifugation (15000 x g, 15 min) [21] . Proteins were This article is protected by copyright. All rights reserved. 6
precipitated by TCA/acetone [22] , washed with acetone, dissolved in rehydration buffer and concentrations were determined by using 2-D Quant Kit (GE Life Sciences).
8
Co-IP of STAT2 and FLAG-tagged Proteins
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For Stat2 co-immunoprecipitation assays, cells were lysed in RIPA buffer composed of 20 mM Tris-HCl pH 7.4, 150 mM NaCl,10 mM CaCl2, 0.1% v/v Triton X-100, 10% v/v glycerol and complete protease inhibitor cocktail (Mini Protease Inhibitor Tablets, Roche). The supernatant was separated by centrifugation at 12.000 g at 4°C for 10 min and incubated with STAT2 antibody (2 µg) (Santa Cruz Biotechnology, 514193) for 3 h at 4°C with gentle shaking. Complexes were precipitated with protein A/G Plus-agarose (Santa Cruz Biotechnology), washed with TBS and resuspended in SDS sample buffer. Immunoprecipitates were subjected to SDS-PAGE and western blotting, as described above.
FLAG-tagged proteins were immunoprecipitated with anti-FLAG M2-agarose (Sigma), following the manufacturer’s instructions.
All assays were performed three times and representative blots are presented.
FLAG-tagged proteins were immunoprecipitated with anti-FLAG M2-agarose (Sigma), following the manufacturer’s instructions.
All assays were performed three times and representative blots are presented.
9
Protein Extraction and Western Blotting
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Cells were washed twice with ice-cold 1 × PBS and lysed using RIPA lysis buffer (Sigma) supplemented with cOmplete, Mini Protease Inhibitor Tablets (1 per 10mL) (Roche) and PhosSTOP phosphatase inhibitor tablets (1 per 10mL). Lysates were sonicated and then centrifuged at 12000 rpm at 4°C for 10 min. Total protein concentration was quantified with pierce BCA protein assay kit (Thermo Scientific™).10 μg of total cell protein were loaded onto 10% SDS-PAGE and then transferred to a microporous polyvinylidene difluoride (PVDF) membrane. Western blotting was performed using Polyclonal anti-GLUT1 antibody (1:2000) (ab652, Abcam), anti-β-actin antibody (Sigma) and goat anti-rabbit or goat anti-mouse IgG-peroxidase conjugate as the secondary antibody. The detection of the proteins was made using chemiluminescence substrate.
10
Immunoblot Characterization of PRKDC-Deficient A549 Cells
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Immunoblotting was used for characterization of A549
PRKDC-/-cells. Cells were lysed using a lysis buffer containing protease inhibitor (Mini Protease Inhibitor Tablets – Roche, 5056489001). The samples were incubated for 30 min at 4 °C, being briefly vortexed each 10 min followed by centrifugation at 13.000 x g for 10 min at 4 °C. The supernatant was transferred to a new tube and the total proteins were quantified using Pierce BCA protein assay kit (Thermo, 23225). From total proteins, 20 µg were transferred to polyacrylamide gel electrophoresis for protein separation and then transferred to nitrocellulose 0.22 µm blotting membranes. The membranes were blocked in 5% non-fat milk in TBS containing 0.1% Tween 20 (TBST) for 1 hour at room temperature. Membranes were then probed with primary antibodies (Supplementary Table IV Supplementary Table V
PRKDC-/-cells. Cells were lysed using a lysis buffer containing protease inhibitor (Mini Protease Inhibitor Tablets – Roche, 5056489001). The samples were incubated for 30 min at 4 °C, being briefly vortexed each 10 min followed by centrifugation at 13.000 x g for 10 min at 4 °C. The supernatant was transferred to a new tube and the total proteins were quantified using Pierce BCA protein assay kit (Thermo, 23225). From total proteins, 20 µg were transferred to polyacrylamide gel electrophoresis for protein separation and then transferred to nitrocellulose 0.22 µm blotting membranes. The membranes were blocked in 5% non-fat milk in TBS containing 0.1% Tween 20 (TBST) for 1 hour at room temperature. Membranes were then probed with primary antibodies (
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