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Tapi 2

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TAPI-2 is a protease inhibitor that can be used in laboratory research settings. It functions as an inhibitor of caspase-2, caspase-3, and caspase-8 enzymes. The core function of TAPI-2 is to inhibit these proteases, which play roles in cellular processes such as apoptosis and inflammation.

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Lab products found in correlation

Human ab serum, by Merck Group (2 mentions) Benzonase, by Merck Group (2 mentions) Sp600125, by Bio-Techne (2 mentions) Sb203580, by Bio-Techne (2 mentions) Neurobasal, by Thermo Fisher Scientific (1 mentions) Penstrep solution, by Corning (1 mentions) Pen strep solution, by American Type Culture Collection (1 mentions) Laminin, by BioLamina (1 mentions) Poly ornithine, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Accutase, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions) Ly294002, by Merck Group (1 mentions) Whitley h35 hypoxystation, by Don Whitley Scientific (1 mentions) Stereotaxic instrument, by Stoelting (1 mentions) Dynasore, by Merck Group (1 mentions) Tnfrsf1a, by Jackson ImmunoResearch (1 mentions) C57bl 6j, by Jackson ImmunoResearch (1 mentions) Duoset elisa kits, by Cloud-Clone (1 mentions) Diphenyleneiodonium chloride, by Merck Group (1 mentions)

15 protocols using tapi 2

1

Thawing and Preparing Cryopreserved Cells

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Cryopreserved samples were thawed in a 37°C water bath and vial contents transferred to a 15 mL Falcon tube. Pre-warmed defrost media (RPMI (Glutamax with HEPES) (Life Technologies (Thermo Fisher)), 5% human AB serum (Sigma), 20 nM TAPI-2 (Sigma), 50 U/mL Benzonase (Sigma) was added dropwise to 10 mL. Cells were rested in 4 mL resting media (RPMI with 10% human AB serum, 20 nM TAPI-2) for 1 hour at 37°C. 2 x 106 (link) cells were used for subsequent flow cytometry staining.
Edner N.M., Heuts F., Thomas N., Wang C.J., Petersone L., Kenefeck R., Kogimtzis A., Ovcinnikovs V., Ross E.M., Ntavli E., Elfaki Y., Eichmann M., Baptista R., Ambery P., Jermutus L., Peakman M., Rosenthal M, & Walker L.S. (2020). Follicular helper T cell profiles predict response to costimulation blockade in type 1 diabetes. Nature immunology, 21(10), 1244-1255.
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2

Thawing and Preparing Cryopreserved Cells

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Cryopreserved samples were thawed in a 37°C water bath and vial contents transferred to a 15 mL Falcon tube. Pre-warmed defrost media (RPMI (Glutamax with HEPES) (Life Technologies (Thermo Fisher)), 5% human AB serum (Sigma), 20 nM TAPI-2 (Sigma), 50 U/mL Benzonase (Sigma) was added dropwise to 10 mL. Cells were rested in 4 mL resting media (RPMI with 10% human AB serum, 20 nM TAPI-2) for 1 hour at 37°C. 2 x 106 (link) cells were used for subsequent flow cytometry staining.
Edner N.M., Heuts F., Thomas N., Wang C.J., Petersone L., Kenefeck R., Kogimtzis A., Ovcinnikovs V., Ross E.M., Ntavli E., Elfaki Y., Eichmann M., Baptista R., Ambery P., Jermutus L., Peakman M., Rosenthal M, & Walker L.S. (2020). Follicular helper T cell profiles predict response to costimulation blockade in type 1 diabetes. Nature immunology, 21(10), 1244-1255.
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3

Glioblastoma Cell Culture Protocol

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PIGPCs were isolated as previously described [39 (link)] and grown in DMEM (Life Technologies) with 10% fetal bovine serum (FBS) and 1% PenStrep solution (Corning). U3046MG, U3035MG, U3082MG, U3084MG, and U3065MG cells were obtained from the Human Glioblastoma Cell Culture Resource (HGCC) and cultured as described previously [23 (link)] in Neurobasal (GIBCO) and DMEM/F12 with Glutamax media (Life Technologies), 1:1 mix, with 1% PenStrep, N2 and B27 (Life Technologies), 10 ng/mL epidermal growth factor (EGF), and 10 ng/mL fibroblast growth factor (FGF) (Peprotech). Cells were dissociated by Accutase (ThermoFisher) treatment, and grown as a monolayer on plastic dishes coated with polyornithine (Sigma) and laminin (Biolamina). U251MG and T98G cells were obtained from ATCC and grown in DMEM with 10% FBS and 1% PenStrep solution. Cells were used within ten passages. Mycoplasma contamination was tested every 3 months.
Hypoxia (1%O2) was generated in a Whitley H35 Hypoxystation (Don Whitley Scientific, Bingley, UK).
Reagents: TAPI-2 (Sigma), MI1 (Tocris Bioscience), LY294002 (Sigma), added 24 h pre-hypoxia exposure.
Grassi E.S., Pantazopoulou V, & Pietras A. (2020). Hypoxia-induced release, nuclear translocation, and signaling activity of a DLK1 intracellular fragment in glioma. Oncogene, 39(20), 4028-4044.
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4

Intracerebroventricular Infusion of TAPI-2 Inhibitor

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The p75NTR metalloprotease inhibitor TAPI-2 (Sigma–Aldrich, St. Louis, MO, USA) was intracerebroventricular injected by the osmotic pump infusion 4 days before 5-bromo-2′-deoxyuridine (BrdU) injection (Figure 1). The mouse was placed in the stereotaxic instrument (Stoelting, Wood Dale, IL, USA), and a 7-day Alzet osmotic minipump (mean flow rate, 0.25 μl/h) containing TAPI-2 (50 mmol/l; Katakowski et al., 2007 (link)) or sterile saline was placed subcutaneously in the back. A brain infusion cannula connected to the pump that was positioned at right lateral ventricle (0.3 mm posterior, 1.0 mm lateral, and 2.3 mm ventral to Bregma).
Qin Y., An D., Xu W., Qi X., Wang X., Chen L., Chen L, & Sha S. (2020). Estradiol Replacement at the Critical Period Protects Hippocampal Neural Stem Cells to Improve Cognition in APP/PS1 Mice. Frontiers in Aging Neuroscience, 12, 240.
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5

Bone Marrow-Derived Macrophage Infection Protocol

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Bone marrow-derived macrophages (BMDMs) from C57BL/6J (Jackson), Tnfrsf1a−/− (23 (link)) (Jackson), Trif−/− (24 (link)) and Trif−/−Tnfr1−/− mice (25 (link)) were cultured and infected as previously described (19 (link)). Y. pseudotuberculosis strain IP2666 and isogenic yopJ mutant bacteria were grown overnight with aeration in 2×YT broth at 26 °C. Yp were diluted into inducing media (2×YT containing 20mM sodium oxalate and 20mM MgCl2) and grown with aeration for 1 h at 26 °C followed by 2 h at 37 °C. BMDMs were infected at a multiplicity of infection (MOI) of 20:1, unless otherwise noted. Cells were incubated at 37 °C and gentamicin (100 μg/mL) was added 1 h after infection. 100 μM zVAD-fmk (zVAD; SM Biochemicals), 60 μM necrostatin-1 (Nec-1; Calbiochem), 3 μM GSK2399872A (GSK’872; GlaxoSmithKline), 50μM TAPI-2 (Sigma), 80μM dynasore (Sigma) were added 1 h before infection where indicated.
Peterson L.W., Philip N.H., Dillon C.P., Bertin J., Gough P.J., Green D.R, & Brodsky I.E. (2016). Cell-extrinsic TNF collaborates with TRIF signaling to promote Yersinia-induced apoptosis. Journal of immunology (Baltimore, Md. : 1950), 197(10), 4110-4117.
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6

ADAM17 Substrate Production Evaluation

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For evaluating the production of ADAM17 substrates, the keratinocytes of scratch wound assay received 24h incubation in medium which did not contain any exogenous EGFR ligands, and were then collected following the process above mentioned 17 (link). Relevant DuoSet ELISA kits (Cloud-clone Corp, AREG SEA006Hu, HB-EGF SEB479Hu, TGF-α SEA123Hu, China) were used to analyze the collected medium specimens regarding the proteins. Supernatants were collected in triplicate for each cell line, and results from the vector and CD9-silenced keratinocytes treated with ADAM17 inhibitor TAPI-2 (Sigma, USA) at final concentration 40 μM.
Liu J., Zhu G., Jia N., Wang W., Wang Y., Yin M., Jiang X., Huang Y, & Zhang J. (2019). CD9 regulates keratinocyte migration by negatively modulating the sheddase activity of ADAM17. International Journal of Biological Sciences, 15(2), 493-506.
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7

ADAM17 Knockdown and Inhibition in Keratinocytes

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On-target siRNA specific to ADAM17 (GenePharma, China) was used to carry out siRNA knockdown on cells under culture. Briefly, penicillin- and streptomycin-free medium, which contained 0.1 μM ADAM17 or non-targeting pool (NTP) siRNA, were used to incubate the keratinocytes for 24 h. Western blot was employed to evaluate ADAM17 expression. Keratinocytes were treated with ADAM17 inhibitor TAPI-2(Sigma CAS 187034-31-7, USA) at final concentration 40 μM and recombinant human heparin-binding EGF (Abcam ab167830, UK) at final concentration 50 mg.
Liu J., Zhu G., Jia N., Wang W., Wang Y., Yin M., Jiang X., Huang Y, & Zhang J. (2019). CD9 regulates keratinocyte migration by negatively modulating the sheddase activity of ADAM17. International Journal of Biological Sciences, 15(2), 493-506.
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8

Lipid Signaling Molecules and Inhibitors

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18:1. lysophosphatidylserine (LysoPS), 18:1 lysophosphatidylcholine (LysoPC), and 18:1 lysophosphatidylethanolamine (LysoPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Epidermal growth factor (EGF) and TGF-α neutralizing antibody were obtained from R&D Systems (Minneapolis, MN, USA). EGFR neutralizing antibody (clone LA1), Z-VAD-fmk, BAY11-7082, diphenyleneiodonium chloride (DPI), N-acetyl-L-cysteine (NAC), TAPI2, suramin, and A438079, Ac-YVAD-cmk were purchased from Sigma-Aldrich (St. Louis, MO, USA). U0126 and AG1478 were purchased from Calbiochem (La Jolla, CA, USA). SB203580 and SP600125 were obtained from Tocris Bioscience (Bristol, UK). PTX, a Gi/o protein inhibitor, was supplied by Invitrogen Life Technologies (Carlsbad, CA, USA). C29 was purchased from Biovision (Milpitas, CA, USA).
Sim M.S., Kim H.J., Jo S.H., Kim C, & Chung I.Y. (2022). Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner. International Journal of Molecular Sciences, 23(7), 3866.
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9

MCF-10A Cells Treated with TAPI-2

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MCF-10A cells were treated for 48 h with 20 µM TAPI-2 (TNF protease inhibitor 2) acetate salt (Sigma-Aldrich, Germany). The culture supernatant was tested for the presence of IL-6 and sIL-6RA by ELISA.
Werner-Klein M., Grujovic A., Irlbeck C., Obradović M., Hoffmann M., Koerkel-Qu H., Lu X., Treitschke S., Köstler C., Botteron C., Weidele K., Werno C., Polzer B., Kirsch S., Gužvić M., Warfsmann J., Honarnejad K., Czyz Z., Feliciello G., Blochberger I., Grunewald S., Schneider E., Haunschild G., Patwary N., Guetter S., Huber S., Rack B., Harbeck N., Buchholz S., Rümmele P., Heine N., Rose-John S, & Klein C.A. (2020). Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency. Nature Communications, 11, 4977.
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10

ADAM17 Regulation of EGFR Signaling

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ADAM17‐small interference RNA (target sequence of 5′‐CCAUGAACACGUGU‐3′) and a negative control (NC) siRNA (target sequence of 5′‐UUACACGUGUUCUUCAUGGT‐3′) were purchased from GenePharma (China). In brief, the cell monolayer was incubated in penicillin‐ and streptomycin‐free culture medium involved in 0.1 μM ADAM17‐siRNA or NC siRNA for 24 hours. Western blot and immunofluorescence were used for evaluating ADAM17 expression. Cell monolayer was treated with ADAM17 inhibitor TAPI‐2 (Sigma CAS 187034‐31‐7, USA) at a final concentration of 40 μM, EGFR inhibitor AG1478 (Calbiochem, La Jolla, CA) at a final concentration of 10 μM and recombinant human heparin‐binding EGF (R&D Systems, Wiesbaden‐Nordenstadt, Germany) at a final concentration of 100 ng/ml.
Jia N., Liu J., Zhu G., Liang Y., Wang Y., Wang W., Chen Y., Yang J., Zhang W, & Zhang J. (2020). Polarization of ADAM17‐driven EGFR signalling in electric field‐guided collective migration of epidermal sheets. Journal of Cellular and Molecular Medicine, 24(23), 14073-14085.
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