Protease inhibitor cocktail set III and phosphatase inhibitor cocktail set V were purchased from Sigma-Aldrich. Where indicated, cells were treated with 10 or 12.5 μM MG132 (Sigma-Aldrich), 3 μM MLN4924 (Sigma-Aldrich), 25 μM P22077 (Sigma-Aldrich), 10 μM FT671 (MedChemExpress, NJ, USA), and 100 nM Bafilomycin A1 (Sigma-Aldrich).
P22077
Manufactured by Merck Group
Sourced in United States, Germany
The P22077 is a laboratory equipment product manufactured by Merck Group. It is a precision instrument designed for specific laboratory applications. The core function of this product is to perform a defined set of tasks within a controlled laboratory environment. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (2 mentions)
Hbx 41 108, by Bio-Techne (2 mentions)
Ubiquitin monomer, by Merck Group (2 mentions)
Pimozide, by Bio-Techne (2 mentions)
Maldi tof ms materials, by Bruker (2 mentions)
Degrasyn wp1130, by Bio-Techne (2 mentions)
L434078, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Pr 619, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Ft671, by MedChemExpress (1 mentions)
Phosphatase inhibitor cocktail set 5, by Merck Group (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Mln4924, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Ab3742, by Abcam (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
Aphidicolin, by Merck Group (1 mentions)
Cisplatin, by Selleck Chemicals (1 mentions)
Hydroxyurea, by Merck Group (1 mentions)
6 protocols using p22077
1
Protein Modulation Inhibitor Protocol
2
Generating Custom Anti-RNF4 Antibody
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Custom anti-RNF4 antibody was generated as previously described (79 (link)). Commercial antibodies used in this study included: SUMO2/3 (ab3742, Abcam), p53 (2524, Cell Signaling Technology [CST]), cleaved caspase-3 (9661, CST), tubulin (T8238, Sigma-Aldrich), KAP1–p-S824 (A300-767A, Bethyl), CHK1–p-S345 (2341, CST), CHK1 (sc-8408, Santa Cruz Biotechnology), γ-H2AX (05-636, Millipore), γ-H2AX–Alexa Fluor 488 (20304S, CST), histone H3 (ab1791, Abcam), TOP2a (sc-365916, Santa Cruz Biotechnology), TOP2b (MAB6348, Novus), PARP1 (9542, CST), RNF10 (16936-1-AP, Proteintech). Antibody specific for mouse POLD4 was provided by Marietta Lee (New York Medical College, Valhalla, New York, USA). The following chemicals were used: olaparib (S1060, Selleckchem), hydroxyurea (H8627, Sigma-Aldrich), aphidicolin (A4487, Sigma-Aldrich), cisplatin (S1166, Selleckchem), MMS (129925, Sigma-Aldrich), gemcitabine (G6423, Sigma-Aldrich), P22077 (662142, Sigma-Aldrich), P5091 (2277, Biovision), 2-D08 (SML1052, Sigma-Aldrich), ML-792 (407886, MedKoo), RI-1 (SML1294, Sigma-Aldrich), and B02 (SML0364, Sigma-Aldrich). For plasmids, human RAD51 cDNA was inserted into pRK5-FLAG, and human RNF4 cDNA was inserted into pMX-PIE-IRES-EGFP. Amino acid substitutions for RNF4-CS and RNF4-ΔSIM were performed as previously described (78 (link), 80 (link)).
3
Cellular Fractionation and Protein Inhibition
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P22077 (Merck-Millipore), NMS873 (Tocris), cycloheximide (Sigma, Merck), ML792 (Synthetized in the CNIO) and MLN7243 (Chemietek) were dissolved in DMSO; cells were incubated for the indicated time in the presence of the inhibitor or an equivalent amount of DMSO. Whole cell extracts were prepared by lysing cells in 50 mM Tris, pH 7.5, 8 M Urea, and 1% Chaps. Cytosolic and nuclear extracts were prepared following the protocol described before (Lecona et al., 2008 (link)) and the chromatin fraction was then extracted in 50 mM Tris, pH 7.5, 8 M Urea, and 1% Chaps. Transfection of RPE cells with specific siRNA was carried out using Lipofectamine RNAimax (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions and using pools of 4 specific siRNA directed against the indicated proteins (Dharmacon, Horizon Discovery). Transfection of HCT116 cells with the pCL-His-hUbi plasmid (Young et al., 2011 (link)) was carried out using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions.
4
Diubiquitin Topoisomer Characterization
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Ubiquitin monomer, BSA, Tris and DTT were purchased from Sigma-Aldrich. Diubiquitin topoisomers (M1, K6, K11, K27, K29, K33, K48 and K63-linked) were purchased from Boston Biochem (Boston, MA), additional K27 diubiquitin was produced in-house57 (link)58 , whereas all MALDI-TOF MS materials (targets, matrix and protein calibration mixture) were purchased from Bruker Daltonics (Bremen, Germany). PR-619 and P22077 (Calbiochem/Merck, Darmstadt, Germany) as well as HBX 41,108, pimozide and degrasyn/WP1130 (Tocris Bioscience, Bristol, UK) and L434078 (Sigma-Aldrich, St Louis, MO, USA) were purchased commercially. Febuxostat, SJB3-019A, compound 16, NSC 697923 and BAY 11-7082 were synthesized (Supplementary Table 3 ).
5
Quantitative Analysis of Diubiquitin Linkages
6
Ubiquitin Deubiquitinase USP7 Regulation
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DUB plasmids and HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA). USP7WT (USP7 wild-type) and USP7C223S (USP7 catalytic mutant) were cloned into a pFLAG-CMV-4 vector. GFP-tagged USP7 constructs (WT or mutants) were kindly provided by Prof. Jing Liu (Central South University, Changsha, China). Additionally, pcDNA3-Myc-ICN1 and pcDNA3-FLAG-ICN1 were obtained from Prof. Hudan Liu (Wuhan University, Wuhan, China).
The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA). All antibodies were diluted according to the manufacturer’s recommendations.
The USP7 inhibitor P22077 [1-(5-((2,4-difluorophenyl)thio)-4-nitrothiophen-2-yl) ethanone] was obtained from EMD Millipore (EMD Millipore, Billerica, MA, USA). MG132 and cycloheximide (CHX) were purchased from Sigma-Aldrich.
The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA). All antibodies were diluted according to the manufacturer’s recommendations.
The USP7 inhibitor P22077 [1-(5-((2,4-difluorophenyl)thio)-4-nitrothiophen-2-yl) ethanone] was obtained from EMD Millipore (EMD Millipore, Billerica, MA, USA). MG132 and cycloheximide (CHX) were purchased from Sigma-Aldrich.
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