Pma Activation Of Jurkat Cells Culture' title='Pma Activation Of Jurkat Cells Culture' />C1.H1. 6O8 Pub. Measurement of Endogenous MALT1 Activity BIO PROTOCOLAbstract.MALT1Mucosa associated lymphoid tissue protein 1 is an important adapter protein for the NF k.B driven lymphocyte activation and the development and survival of distinct B cell lymphoma entities.In addition MALT1 is a cysteine protease that structurally resembles caspases while having a different substrate preference and mechanism of activation.This paracaspase activity of MALT1 has been shown to be critical for an optimal NF k.B activation and survival of the aggressive ABC DLBCL Activated B cell type of diffuse large B cell lymphoma, which highlights the protease as an attractive therapeutic target for the treatment of distinct B cell lymphomas and immune diseases like rheumatoid arthritis or multiple sclerosis.In this protocol we describe a fluorogenic cleavage assay, which can be used to measure endogenous and also ectopic MALT1 activity.To this end, cellular MALT1 needs to be precipitated from the lysed cells via antibody immunoprecipitation and subsequently incubated with a fluorogenic substrate peptide.The MALT1 cleavage assay has been developed to directly determine the activity profile of MALT1 in the course of the adaptive immune response as well as in pathological signaling in lymphoid malignancies.In addition, the MALT1 activity assay has been successfully used to monitor cellular MALT1 inhibition with small molecule inhibitors.Keywords Protease, Paracaspase, Immune signaling, T cell activation, Lymphoma.Materials and Reagents.Primary human and murine cells.T cell lines Jurkat.B cell lines ABC TMD8, HBL1, OCI Ly.U2. 93. 2, OCI Ly.SigmaAldrich offers SigmaP1585, PMA for your research needs.Find product specific information including CAS, MSDS, protocols and references.RIVA and GCB DLBCL Su DHL 6, Su DHL 4, BJAB.RPMI 1. 64. 0 Life Technologies, catalog number 2.IMDM Life Technologies, catalog number 2.Fetal Bovine Serum FBS Life Technologies, catalog number 1.F1.large.jpg' alt='Pma Activation Of Jurkat Cells Culture' title='Pma Activation Of Jurkat Cells Culture' />PMA Phorbol 1.Myristat 1. Acetat Calbiochem, catalog number 1.Lonomycin Calbiochem, catalog number 4.Anti CD3CD2. 8 Hit.CD2. 8. 2 BD Heidelberg.F6.medium.gif' alt='Pma Activation Of Jurkat Cells Culture' title='Pma Activation Of Jurkat Cells Culture' />Ig.G1 R1. 9 1. BD Pharmingen, catalog number 5.Ig. G2a A8. 5 1 BD Pharmingen, catalog number 5.MALT1 antibody Santa Cruz, catalog number H3.Protein G Sepharose GE Healthcare, catalog number 1.PBS Life Technologies, catalog number 1.Ac LRSR AMC Peptides International, catalog number MCA 3.PI. Z VRPR FMK ENZO Life Sciences, catalog number ALX 2.Complete protease inhibitor cocktail tablets Roche, catalog number 1.Mepazine Hit. 2lead, catalog number 5.Greiner Bio one, catalog number 7.Cellular lysis buffer see Recipes.MALT1 cleavage buffer see Recipes.C 5 CO2 cell culture incubator.G syringe Roth, catalog number C7.Centrifuge Eppendorf, model 5.R. Synergy II multiwell plate reader Biotek.To activate MALT1 plate 2.Jurkat T cells, primary T cells or PBMCs and either stimulate with PMAIonomycin 2.RPMI media in 2. 5 cm.CD3 and anti CD2.Ig. G1Ig. G2a coupling antibodies 0.RPMI media in 1. 2 well for 3.H2. O in the control.Cells with constitutive MALT1 activity e.ABC DLBCL can be left untreated.As a negative control treat the above cells with MALT1 inhibitors, e.Z VRPR FMK 5. 0 M or small molecule inhibitors like mepazine 1.M 3 6 h prior to stimulation and lysis.All 2. 5 x 1. 06 cells per reaction are then pelleted at 3.Removal of supernatant and lysis of the cells with cellular lysis buffer 5.C in a 1. 5 ml reagent tube. Install Windows Xp Step By Step Instructions . Centrifugation of the lysates for 1.Incubation of all of the lysate with 7.MALT1 antibody over night at 4 C on a rotary mixer.Incubation with 1.Protein G sepharose beads beads were washed and diluted 1 2 in PBS and equilibrated before usage for 6.C on a rotary mixer.Beads are pelleted and washed 3 times with PBS at 3.C. In the last washing step all PBS is discarded via a syringe and 4.The beads are pelleted at 3.Ac LRSR AMC is then added to the wells in a final concentration of 2.M and the plates are placed into the plate reader where they are shaked for 1.After an incubation of 3.C the release of AMC fluorescence due to MALT1 cleavage is measured at 3.Specificity of MALT1 cleavage activity in vitro can be assessed by adding 5 n.M of Z VRPR FMK to a separate control reaction negative control.Figure 1. Fluorogenic MALT1 cleavage assay.Jurkat T cells 2.Mepazine or DMSO for 4 h and subsequently stimulated with PMAIonomycin PI or anti.CD3CD2. 8 for 3. After lysis of the cells MALT1 was precipitated and the catalytic activity was measured after addition of Ac LRSR AMC in a Synergy II plate reader over 6.Cellular lysis buffer.M HEPES p. H 7. 51.Glycerin 0. 1 vvTriton X 1.M Dithiothreitol DTT1.M Na. Cl. 2 m. M Mg.Cl. 21 complete protease inhibitor tablet per 5.MALT1 cleavage buffer.M MES p. H 7. 01.M Na. Cl. 10 wv Saccharose 0.CHAPS 1 M Sodium citrate.M DTT. Acknowledgments.This protocol was adapted from two previous publications Kloo et al., 2.Nagel et al., 2. 01.The work was supported by a grant of the Deutsche Krebshilfe e.V. to DK. Kloo, B., Nagel, D., Pfeifer, M., Grau, M., Duwel, M., Vincendeau, M., Dorken, B., Lenz, P., Lenz, G.Krappmann, D. 2. Critical role of PI3.K signaling for NF kappa.B dependent survival in a subset of activated B cell like diffuse large B cell lymphoma cells.Proc Natl Acad Sci U S A 1.Nagel, D., Spranger, S., Vincendeau, M., Grau, M., Raffegerst, S., Kloo, B., Hlahla, D., Neuenschwander, M., Peter von Kries, J., Hadian, K., Dorken, B., Lenz, P., Lenz, G., Schendel, D.J. and Krappmann, D.Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC DLBCL.Cancer Cell 2. 26 8.Copyright 2. 01.The Authors exclusive licensee Bio protocol LLC.How to cite Nagel, D.Krappmann, D. 2. Measurement of Endogenous MALT1 Activity.Bio protocol 31. DOI 1.Bio. Protoc. 8. 21.
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