Isolation of B cells from spleen or peripheral lymph nodes was performed by using the B Cell Isolation Kit from Miltenyi Biotec. B cells were cultured in RPMI 1640 Medium (Dutch Modification) plus 5% FCS, antibiotics, 2 mM L-glutamine and β-mercaptoethanol (5 µM). Etoposide (Sigma Aldrich, 20 µM) was added to the B cell cultures after 48 h stimulation with either LPS (Sigma Aldrich, E.coli 0127:B8, 10 µg ml−1) or with an anti-CD40 antibody (3/23 clone, 10 µg ml−1) plus recombinant mouse IL-4 (Peprotech, 10 ng ml−1) and recombinant mouse IL-5 (Sigma Aldrich, 5 ng ml−1). KU55933 (ATM inhibitor, Tocris, 10 µM), SB203580 (p38 inhibitor, Cell Signalling, 10 µM), Chk2 inhibitor II (Chk2 inhibitor, Sigma Aldrich, 10 µM), and AZD7762 (Chk1/2 inhibitor, Axon MedChem, 10 µM) were added, when indicated, 1-h prior treatment with Etoposide. Actinomycin D (ActD, 5 µg ml −1), cyclohexamide (CHX, 100 μg ml−1), MG132 (10 μM), Puromycin and SJ172550 were purchased from Sigma Aldrich. HBX41108 and P005091 were purchased from Tocris. Lactacystin, Pifithrin-α, Nutlin-3, p53 Activator III RITA and 4EGI-1 were puchased from Calbiochem.
Hbx 41 108
Manufactured by Bio-Techne
Sourced in Germany
The HBX 41,108 is a laboratory equipment product manufactured by Bio-Techne. It is designed to perform a specific function, but a detailed and unbiased description cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
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)
P22077, by Merck Group (2 mentions)
Ku55933, by Bio-Techne (1 mentions)
Recombinant mouse il 4, by Thermo Fisher Scientific (1 mentions)
Cyclohexamide chx, by Merck Group (1 mentions)
E coli 0127 b8, by Merck Group (1 mentions)
Sj172550, by Merck Group (1 mentions)
B cell isolation kit, by Miltenyi Biotec (1 mentions)
Mg132, by Merck Group (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
Puromycin, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Etoposide, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
7 protocols using hbx 41 108
1
B Cell Isolation and Etoposide Treatment
2
Antibody Sources and Reagents for Protein Analysis
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The sources of antibodies against the following proteins were as follows: β-actin (A1978) and FLAG (F3165) from Sigma; ECT2 (07-1364) from Millipore; PHF8 (A301-772A) and USP7 (A300-033A, for WB, IF, IP and IHC) from Bethyl Lab; USP7 (05-1946 for WB) from Sigma; RNF168 (21393-1-AP), USP11 (10244-1-AP), RAD18 (18333-1-AP), UHRF1 (21402-1-AP), and His (66005-1-Ig) from Proteintech; Myc (M047-3) from MBL; MDM2 (ab38618, WB and IHC), CDC42 (ab187642), RAC1 (ab33186) from Abcam; GFP (YM3124) from Immunoway; and ubiquitin (OM294553) from OmnimAbs. Anti-FLAG M2 affinity gel (A2220), 3 × FLAG peptide (F4799), MG132 (SML1135), neomycin (N1142), blasticidin (15205), puromycin (P8833) and doxycycline (D9891) were purchased from Sigma. Ni-NTA Purification System (K950-01) was purchased from Thermo Fisher. K48-linked tetra-ubiquitin chains (UC-210B) and K63-linked tetra-ubiquitin chains (UC-310B) were purchased from Boston Biochem. CHX and HBX 41,108 were purchased from TOCRIS. GNE-6640 was purchased from Glixx Laboratories.
3
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 ).
4
Comparative Analysis of hASCs and hBMMSCs
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Primary hASCs and human bone marrow-derived mesenchymal stem cells (hBMMSCs) were purchased from ScienCell Company (San Diego, CA, USA). Stem cells used in the cell-based experiments were collected from three donors, and all the in vitro experiments were repeated in triplicate. All materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless specially mentioned. Cells were cultured in proliferation media (PM) consisting of Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), 10% fetal bovine serum (ScienCell), and 100 IU/ml penicillin/streptomycin (Gibco). Osteogenic differentiation of cells was induced when cells grew to 80–90% confluence with osteogenic media (OM) containing standard PM supplemented with 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate. Adipogenic differentiation of cells was induced when cells grew to 100% confluence with adipogenic media (AM) containing standard PM supplemented with 10 μM insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 200 μM indomethacin. HBX 41,108 was purchased from Tocris (MN, USA).
5
USP7 Inhibitor Protocols Synthesis
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P5091, MG132 and Bortezomib were purchased from SelleckChem. P22077 and HBX41108 were obtained from TOCRIS. MLN7243 was purchased from Chemietek. Bafilomycin A1 was obtained from MilliporeSigma. The rest of known USP7 inhibitors, WNT974, and TNKS656 were synthesized according to published literature procedures by Novartis.
6
Quantitative Analysis of Diubiquitin Linkages
7
Inhibitors for USP7 and USP30 Enzymes
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USP7 inhibitors FT671 and FT827 (Ioannidis et al., 2016 ; Turnbull et al., 2017 (link)) were a kind gift from Stephanos Ioannidis. USP30 inhibitor 39 (Kluge et al., 2018 (link)), and USP30 inhibitor 3-b (patent WO2020072964) were kindly provided by Jeff Schkeryantz and Lixin Qiao (Evotec/Bristol-Meyers-Squibb). Inhibitor structures are shown in Supplementary Figure S3 . PR619 was purchased from Calbiochem (Cat. No. 662141), N-Ethylmaleimide from Sigma-Aldrich (Cat. No. E3876), USP7 inhibitor P22077 from Calbiochem (Cat. No. 662142), and USP7 inhibitor HBX41108 from TOCRIS (Cat. No. 4285).
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