B ap15

Manufactured by Cayman Chemical

B-AP15 is a small molecule that functions as a proteasome inhibitor. It targets the 20S proteasome subunit and blocks its enzymatic activity.

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4 protocols using b ap15

Latency Reactivation Screening Protocol

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50,000 J-Lat 10.6 and JNLGFP cells were aliquoted into 96-well plates in 200 μL medium. Each of the following drugs were added to the cells in triplicate at the indicated concentrations: BMH-21 (MedKoo, 406586), RA190 (Calbiochem, 5.30341.0001), ERW1041E (Sigma, 509522), ZDON (Calbiochem, 616467), ZDON (Calbiochem, 15227), tubacin (Sigma, SML0065), b-AP15 (Cayman chemical, 11324; AdooQ Bioscience, A15391; MedKoo, 406452), capzimin (Glix labs, GLXC-09966), thiolutin (MedKoo, 525293; Cayman chemical, 11350), WP1130 (Cayman chemical, 15227), P005091 (MedKoo, 406577; Cayman chemical, 15224), IU1 (AdooQ, A13209; Cayman chemical, 10617), TCID (Cayman chemical, 16353; Sigma, SML1402), acitretin (Sigma, 44707), prostratin (Sigma, P0077), ingenol (Sigma, SML1318), vorinostat (Sigma, SML0061), JQ1 (Sigma, SML1524), I-BET151 (Tocris, 4650), 5-azacytidine (Sigma, A3656), romidepsin (Sigma, SML1175), PR619 (AdooQ Bioscience, A13190). For control groups, 0.1% DMSO was used. After 48 or 72 h of treatment, GFP expression in the cells were measured by flow cytometry. Cell viabilities were determined by forward scatter vs. side scatter gating using untreated cells as the control. The data were analyzed with FlowJo software and plotted as bar graphs.

Rathore A., Iketani S., Wang P., Jia M., Sahi V, & Ho D.D. (2020). CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models. Scientific Reports, 10, 5350.

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Molecular Interplay of Ubiquitin Proteases

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bAP15 (11324) was purchased from Cayman Chemical (Ann Arbor, MI). The USP14 inhibitor IU1 (662210) was obtained from EMD Millipore Corporation (Billerica, MA). UCHL5 shRNA plasmid (h) (sc-76797-SH) was purchased from Santa Cruz Biotechnology (Dallas, TX). HA-ubiquitin (Plasmid 18712) was purchased from Addgene (Cambridge, MA).
Antibodies were obtained as follows; anti-β-actin (sc-47778), anti-UCHL5 (sc-271002), anti-USP14 (sc-100630), anti-β-catenin (sc-7963), anti-Vimentin (sc-6260), anti-Ub (sc-166553), and anti-HA-probe (F-7) (sc-7392) (Santa Cruz Biotechnology), anti-E-cadherin (610181), anti-N-cadherin (610920)(BD Bioscience, San Jose, CA), anti-Smad2 (5339), anti-Phospho-Smad2 (3108), anti-Phospho-Smad2 (18338), anti-Smad3 (9523), anti-Phospho-Smad3 (9520), and anti-Smad4 (38454) (Cell Signaling Technology, Danvers, MA).

Fukui S., Nagasaka K., Miyagawa Y., Kikuchi-Koike R., Kawata Y., Kanda R., Ichinose T., Sugihara T., Hiraike H., Wada-Hiraike O., Sasajima Y, & Ayabe T. (2019). The proteasome deubiquitinase inhibitor bAP15 downregulates TGF-β/Smad signaling and induces apoptosis via UCHL5 inhibition in ovarian cancer. Oncotarget, 10(57), 5932-5948.

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Cytokine Signaling Pathway Reagents

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Different reagents and their sources used in this study were: ATP, nigericin, BSA fatty acid-free and low endotoxin, palmitic acid, doxycycline, MCC950 (CP-456773), and the authentic standards citric acid, malic acid, fumaric acid, succinic acid, ketoglutaric acid, pyruvic acid and lactic acid were all purchased from Sigma-Aldrich; palmitic acid was conjugated with BSA as described previously^{58 (link)}; ultrapure E. coli LPS serotype 0111:B4 (tlrl-3pelps) and Pam3CSK₄ (tlrl-pms) from InvivoGen; recombinant mouse IL-1Ra protein (HY-P72566) from MedChemExpress; recombinant mouse IL-6 protein (RMIL6I) from Invitrogen; recombinant mouse S100A9 protein (2065-s9), recombinant human S100A9 protein (9254-s9), recombinant human IL-6 protein (7270-IL/CF) from R&D systems; Pyruvate (L064-100) from Biowest; 2-deoxy-D-glucose (2-DG) (14325), ubiquitin isopeptidase inhibitor I G5 (21006), PR-619 (16276) and b-AP15 (11324) from Cayman Chemical Company. Acetonitrile and water 0.1% (v/v) formic acid were from J,T. Baker, and formic acid was obtained from Panreac. Ultrapure water filter through Milli-Q system (Millipore Corp).

Molina-López C., Hurtado-Navarro L., García C.J., Angosto-Bazarra D., Vallejo F., Tapia-Abellán A., Marques-Soares J.R., Vargas C., Bujan-Rivas S., Tomás-Barberán F.A., Arostegui J.I, & Pelegrin P. (2024). Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production. Nature Communications, 15, 1096.

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Apoptosis Induction Pathway Dissection

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b-AP15 was purchased from Cayman Chemical Company (Ann Arbor, MI). CFZ was purchased from Selleck Chemicals (Houston, TX). BTZ was originally provided by Millennium Pharmaceuticals (Cambridge, MA). CQ was purchased from Sigma Chemical Co. (St. Louis, MO). Baf A was purchased from LC laboratories (Woburn, MA). Human recombinant TRAIL was purchased from PeproTech, Inc. (Rocky Hill, NJ). AMG655 was supplied by Amgen, Inc (Thousand Oaks, CA). Other antibodies were the same as described previously^{45 (link)}.

Oh Y.T., Deng L., Deng J, & Sun S.Y. (2017). The proteasome deubiquitinase inhibitor b-AP15 enhances DR5 activation-induced apoptosis through stabilizing DR5. Scientific Reports, 7, 8027.

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