After transfection of the indicated constructs or siRNAs, HeLa cells stably expressing NLS-BirA and Avi-H2AX were treated with 2 μg ml−1 of biotin (Sigma) overnight, and then collected 1 h after 10 Gy IR. For endogenous USP1 ubiquitination assay or the interaction between Cdh1 and Emi1, HeLa cells were transfected with the indicated siRNAs and various expression constructs. The transfected cells were synchronized with double thymidine block and released into fresh media for 6–8 h when they had reached S/G2 (the cell cycle progression was monitored with FACS analysis). In USP1 ubiquitination experiment, MG132 (10 μM) was added for 6 h during the release. The cells were then irradiated with 10 Gy and collected 1 h after the irradiation. The collected cells were lysed in 100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 6 M Urea and 0.5% SDS (lysis buffer) plus phosphatase (GenDEPOT) and protease inhibitors (Roche) as well as 50 μM PR-619 (a pan-DUB inhibitor, Millipore). After sonication, the supernatants were diluted in lysis buffer without SDS and incubated with Streptavidin-Agarose Slurry (Thermo Scientific) or USP1 antibody-conjugated to protein G beads for 6 h at 4 °C. The agarose beads were then spun down, washed with the lysis buffer (without SDS), and prepared for Western blot analysis.
Pr 619
Manufactured by Merck Group
Sourced in United States, Germany
PR-619 is a piece of laboratory equipment manufactured by Merck Group. It is designed to perform specific functions in a research or laboratory setting. The core function of PR-619 is to [core function description]. Further details about the intended use or interpretation of the product's capabilities are not available.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (9 mentions)
Dithiothreitol (dtt), by Merck Group (4 mentions)
Anti flag antibody, by Merck Group (3 mentions)
1 10 phenanthroline, by Merck Group (3 mentions)
Trichostatin a tsa, by Merck Group (2 mentions)
Curcumin, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Laemmli buffer, by Bio-Rad (2 mentions)
Primary v5 antibody, by Thermo Fisher Scientific (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Lenti x 293t cells, by Takara Bio (2 mentions)
Mln4924, by Abcam (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Mg132, by Cayman Chemical (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Hbx 41 108, by Bio-Techne (2 mentions)
Ubiquitin monomer, by Merck Group (2 mentions)
34 protocols using pr 619
1
Biotin-based Chromatin Immunoprecipitation
2
Modulating Protein Modifications in Cells
3
Modulating Protein Modifications in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to accumulate SUMOylated proteins, cells were treated with 10 μM MG-132 (Sigma) dissolved in DMSO for 7 hours, treated with 20 μM PR-619 (Millipore) dissolved in DMSO for 7 hours, or incubated at 43°C for 1 hour (heat shock). For increasing acetylation of histones, Trichostatin A (TSA, Sigma) was used at a concentration of 150 nM or 600 nM for 18 hours 39 (link). In order to decrease acetylation of histones, curcumin (Sigma) was used at a concentration of 25 μM and 50 μM for 18 hours 39 (link). For transfection, cells were cultured in DMEM lacking penicillin and streptomycin. Transfections were performed using 2.5 μg of polyethylenimine (PEI) per 1 μg of plasmid DNA, using 1 μg of DNA per 1 million cells. Transfection reagents were mixed in 150 mM NaCl and incubated for 15 minutes before adding it directly to the cells. Cells were split after 24 hours and investigated after 48 hours. Lentiviruses were generated essentially as described previously 49 . Infections were performed with a multiplicity of infection of 2 and using a concentration of 8 μg per mL polybrene in the medium. 24 hours after infection the medium was replaced.
4
Inhibition of Proteasome and Deubiquitination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteasome and deubiquitination activity were inhibited in the most mature (largest) leaves of wild type and amiNAA10 plants by floating leaf discs (4 mm) on ½ × Hoagland medium supplemented with 50 μM of MG132 (Santa-Cruz Biotechnology) and 20 μM deubiquitinase inhibitor PR-619 (Sigma Aldrich) for 5 h. After incubation, samples were frozen in liquid nitrogen. Proteins were extracted in 20 mM sodium phosphate buffer pH 7.2, 1% Tween 20, 10 mM DTT, 0.5 mM PMSF, cOmplete protease inhibitor cocktailTM (Roche), and 20 µM PR-619. The resulting crude extract was clarified by centrifugation (30 min at 4 °C, 15000 g) and incubated with 40 µl beads (Ubi-Qapture Q matrix, Enzo life Sciences) overnight. The proteins were eluted from the matrix according to manufacturer’s instructions. Same volume of the different elution fractions was separated by SDS-PAGE and visualized by silver staining (GE Healthcare). The immunological detection was done with the antibody mono- and polyubiquitinylated conjugated to HRP (FK2, HRP conjugate, Enzo life Sciences) provided with the Ubi-Qapture Q kit.
5
Antibody-based Western Blotting and Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for Western blotting or immunoprecipitation: anti-SF3B1 (Bethyl Laboratories, A300-996A-T, MBL International, D221-3), rabbit anti-cleaved Notch1 (Cell Signaling Technology, 4147S), rabbit anti–β-actin (Cell Signaling Technology, 4967S), rabbit anti-USP7 (Bethyl Laboratories, A300-033A), rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling Technology, 2118S), rabbit anti-CHEK2 (Bethyl Laboratories, A300-618A), mouse immunoglobulin G2b (IgG2b) (MBL International, M077-3), P5091 (Selleck Chemicals, S7132), PR619 (Sigma-Aldrich, SML0430), b-AP15 (Selleck Chemicals, S4920), ML323 (Sigma-Aldrich, SML1177), BML277 (MedChem Express, HY-13946), BML277 (Sigma-Aldrich, C3742, in vivo), SCH900776 (MedChem Express, HY-15532), camptothecin (Selleck Chemicals, S1288), flavopiridol (Sigma-Aldrich, F3055), topotecan (Sigma-Aldrich, T2705), etoposide (Sigma-Aldrich, E1383), mitoxantrone (European pharmacopoeia reference standard, M2305000), doxorubicin (Sigma-Aldrich, 44583), and E7107 and H3B-8800 (H3 Biomedicine).
6
Cultivation of Cell Lines and Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human OS (HOS, KHOS, G292, MG63, U2OS, 143B, SAOS2) cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Lonza, Basel, Switzerland) with 10% fetal bovine serum (Hyclone Perbio, Bezons, France) and 1% penicillin–streptomycin (Lonza, Basel, Switzerland). Human mesenchymal stem cells (MSCs) were provided from the Institute for Clinical Transfusion Medicine and Immunogenetics (Ulm, Germany). This center has a production license for MSCs from bone marrow aspirates (production license DE-BW-01-MIA-2013-0040/DE-BW-01-IKT Ulm) using Good Manufacturing Practices according to defined standard operating procedures and in compliance with the established quality management system. MSCs were cultured in Minimum Essential Medium Alpha (1X) (Life technologies, Carlsbad, CA, USA) with 5% platelet lysate plasma, 1 IU/mL heparin sodium (Panpharma, Luitré, France) and 1% penicillin–streptomycin (Lonza). Mycoplasma level has been tested according to the manufacturer’s protocol (Lonza). PR619 was purchased from Sigma (St. Quentin Fallavier, France).
7
Immunoprecipitation of GFP Fusion Proteins
8
Molecular Mechanisms of HBx Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Construction of the eukaryotic expression vectors for wild-type HBx and shRNA against HBx were as described previously23 (link)37 (link). Full length human RPS7 gene was amplified and cloned in pCMV-Tag2b vector with N-terminal FLAG tag. Myc-ubiquitin construct was kindly provided by Dr. Michael MC Lai (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan). Antibodies were obtained from following sources: SIRT7 and H3K18ac from Millipore, HBx, actin, GAPDH, histone H3, ubiquitin, Myc, CBP and p300 antibodies from Santa Cruz Biotechnology, RPS7 antibody from Proteintech and anti-FLAG antibody from Sigma-Aldrich. Seakem-LE agarose used for colony formation assay was obtained from Lonza. Inhibitors PR-619 and MG-132 and MTT used for cell survival assay were purchased from Sigma-Aldrich. Cycloheximide was procured from Calbiochem. Control and SIRT7-specific siRNA were purchased from Invitrogen. For quantitative-PCR, iTaq™ Universal SYBR® Green Supermix from Biorad was used. For immuno-histochemistry, Lab Vision DAB Quanto detection system was procured from Thermo Scientific.
9
Cellular Bead Internalization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PDL-coated beads were prepared at the experiment day. 4.5 μm-diameter aliphatic amine latex beads (Life Technologies) were incubated with PDL for 30 min at 37°C, washed twice in sterile mQH2O and diluted in culture medium or HEPES-buffered solution imaging medium (119 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, 10 mM HEPES, pH 7.4) for experiments requiring fixation or live imaging, respectively. Beads were added to cultures for the indicated periods of time and incubated at 37°C. Drug treatment [IU1 (75 μM, Tocris Bioscience), MG132 (1μM, Calbiochem), PR619 or Ziram (1μM, Sigma Aldrich)] was performed in conditioned medium (either culture or imaging medium) by diluting the drug from a 1000×-concentrated stock in DMSO. Equal amounts of DMSO were added to the control condition. Ziram was always prepared fresh before experiment.
10
Calcium-Induced Keratinocyte Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 48 h the medium was replaced with DFK medium composed of 1:1 of calcium-free Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 mg D-glucose per ml, and Ham’s F12 nutrient mix (Gibco™, Thermo Fisher Scientific, Cat#11765054), supplemented with 0.2 ng EGF per ml, 25 μg BPE per ml, 2 mM L-glutamate (Sigma Aldrich, Cat#G7513-100 ML), 100 unit penicillin, 100 μg streptomycin per ml and 1.5 mM CaCl2 (to trigger calcium induced differentiation; i.e., “calcium switch”). Upon differentiation (>24 h at 1.5 mM CaCl2) mediated by calcium switch, keratinocytes were treated with 10 μM MG132 (Sigma Aldrich, Cat#474790); a potent, reversible proteasome inhibitor or 10 µM PR-619 (Sigma Aldrich, Cat#SML0430-1 MG), a broad range deubiquitinase inhibitor; dissolved in DMSO for 2 h, 4 h, 8 h and 16 h. Protein extraction was performed after 48 h of calcium switch.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!