Anti-RHBDL2 and anti-E-cadherin antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-VE-cadherin (clone BV-9) and anti-HA (clone F-7) antibodies were supplied by Santa Cruz Biotechnology. EGFR phosphorylation was detected by a phospho-specific antibody (directed against p-Tyr1068) from Abcam (Cambridge, UK). The total and phosphorylated forms of p44/42 MAPK(Erk1/2) and AKT (against pAKT-S473 and pMAPK-Thr202/Tyr204) were detected with antibodies from Cell Signaling Technology (Danvers, MA, USA). Other antibodies that were applied in this study were: for Western Blotting, anti-vinculin (Sigma), anti-N-cadherin (Abcam), anti-Thrombomodulin (D-3, Santa Cruz Biotechnology, Dallas, CA, USA), and anti-RFP (Rockland Immunochemicals Inc., Limerick, PA, USA), for immunofluorescence anti-E-cadherin (clone 36/E, BD Transduction Laboratories) and anti-VE-cadherin (clone F-8, Santa Cruz Biotechnology). The secondary antibodies were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The E-cadherin function-blocking antibody DECMA-1 was purchased from Sigma Aldrich. Santa Cruz Biotechnology supplied the IKK inhibitor IKK-16, while the metalloproteases inhibitors BB94 and Marimastat and the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) were from Sigma. Human recombinant TNFα was purchased from Peprotech (Rocky Hill, NJ, USA).
3 4 dichloroisocoumarin dci
Manufactured by Merck Group
Sourced in United States
3,4-dichloroisocoumarin (DCI) is a synthetic organic compound commonly used as a laboratory reagent. It functions as an effective serine protease inhibitor, capable of interfering with the catalytic activity of various enzymes. This compound is primarily utilized in biochemical research and assay development applications.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by Santa Cruz Biotechnology (2 mentions)
Pd98059, by Santa Cruz Biotechnology (2 mentions)
Nf κb inhibitor bay 11 7082, by Enzo Life Sciences (2 mentions)
Ac devd cho, by Enzo Life Sciences (2 mentions)
Cathepsin b inhibitor ca074, by Apexbio (2 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti ha clone f 7, by Santa Cruz Biotechnology (1 mentions)
Ikk 16, by Merck Group (1 mentions)
Marimastat, by Merck Group (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Decma 1, by Merck Group (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Anti rfp, by Rockland Immunochemicals (1 mentions)
P egfr, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 fitc apoptosis kit, by Merck Group (1 mentions)
Ag1478, by Merck Group (1 mentions)
P fak, by Abcam (1 mentions)
Concanamycin a cma, by Merck Group (1 mentions)
Ac yvad cho, by Enzo Life Sciences (1 mentions)
5 protocols using 3 4 dichloroisocoumarin dci
1
Comprehensive Antibody Characterization for Cell Signaling
2
Antibody Detection of Apoptosis Markers
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Specific antibodies against activated (cleaved) caspase 3, phosphorylated focal adhesion kinase (p-FAK), and RHBDL2 were purchased from Abcam (Abcam, Cambridge, MA). Antibodies against epithelial growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), and α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All shRNA expression vectors were purchased from the National RNAi Core Facility Platform of Academia Sinica (Taiwan). The efficient target sequence of RHBDL2 is CCCTTGGAAATGGTCCACAAA. The rhomboid serine protease inhibitor 3,4-dichloroisocoumarin (DCI), EGFR inhibitor AG1478, and the Annexin V-FITC apoptosis kit were purchased from Sigma-Aldrich, Co.
3
Effector Cell Cytotoxicity Assay
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The effect of perforin or serine protease inhibitors on the cytotoxic activity of each effector cell population (CD8 + cells and CD8 -cells) was examined using concanamycin A (CMA, Sigma, St. Louis MO) or 3,4-dichloroisocoumarin (DCI, Sigma, St. Louis MO, USA), respectively. Each effector cell was separated from the gill and kidney of ginbuna carp as described above. Effector cells were treated with CMA at two concentrations (1 and 0.5 µM) for 2 h or DCI at two concentrations (40 and 20 µM) for 3 h at 22°C. The concentrations of these inhibitors were determined according to previous studies, and untreated effector cells were used as a control (Toda et al., 2011a (Toda et al., , 2011c)) . The inhibitortreated effector cells (9.0 × 10 4 cells) were then co-cultured with 300 I. multifiliis cells per well in 96-well plates in the presence of inhibitors for 2 h at 22°C. Dead parasites were then counted in each treated group at all inhibitor concentrations, and the percentage of dead cells was then calculated using the above formula, accounting for negative controls. The percentage of killing activity was calculated as described.
4
Neutrophil Response to Uropathogenic E. coli
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Human neutrophils were stimulated with the wild type CFT073 or CFT073 mutant bacteria for 3 or 6 h at a multiplicity of infection (MOI) of 1 or 10 and incubated at 37 °C with 5% CO2. Neutrophils were also pre-incubated with caspase-1/4 inhibitor AC-YVAD-CHO (10 µM, Enzo Life Sciences, NY, USA), caspase-3 inhibitor AC-DEVD-CHO (10 µM, Enzo Life Sciences), NLRP3 inhibitor MCC950 (2 µM, Avistron Chemistry Services, Cornwall, UK), JNK inhibitor SP600125 (10 µM, InSolutionT M JNK Inhibitor II, Calbiochem, USA), p38 MAPK inhibitor SB203580 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ERK1/2 inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc), NF-κB inhibitor BAY 11–7082 (5 µM, Enzo Life Sciences), serine protease inhibitor 3,4-Dichloroisocoumarin (DCI, 100 µM, Merck Millipore, MA, USA), cathepsin B inhibitor CA074 (100 µM, Apexbio Technology LLC, Houston, USA), actin polymerization inhibitor cytochalasin D (Cyto D, 10 µg/ml, Santa Cruz Biotechnology Inc), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) inhibitor GSK-872 (10 µM, R&D Systems, Minneapolis, USA) or DMSO as vehicle control, for 1 h prior to CFT073 stimulation for 3 or 6 h at MOI 1 or MOI 1015 (link). mRNA, proteins and supernatants were collected and kept at − 80 °C until further analysis.
5
Modulation of Renal Cell Responses to Uropathogenic E. coli
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The renal fibroblasts and the renal epithelial cell lines HK-2 and A498 cells were stimulated with CFT073 wild-type and mutants at a multiplicity of infection (MOI) of 1 or 10 for 6 h and incubated at 37 °C with 5% CO2. The fibroblasts were also pre-incubated with DMSO (vehicle), p38 MAPK inhibitor SB203580 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ERK1/2 inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc.), NF-κB inhibitor BAY 11-7082 (5 µM, Enzo Life Sciences, Farmingdale, NY, USA) and the PKC inhibitor bisindolylmaleimide I (10 µM, Santa Cruz Biotechnology Inc.), JNK inhibitor SP600125 (10 μM, InSolutionT M JNK Inhibitor II, Calbiochem, San Diego, CA, USA), serine protease inhibitor 3,4-Dichloroisocoumarin (DCI, 100 μM, Merck Millipore, MA, USA), cathepsin B inhibitor CA074 (100 μM, Apexbio Technology LLC, Houston, TX, USA), NLRP3 inhibitor MCC950 (2 μM, Avistron Chemistry Services, Cornwall, UK), Disulfiram (10 μM, Selleckchem, Houston, TX, USA), caspase-3 inhibitor AC-DEVD-CHO (10 μM, Enzo Life Sciences) for 1 h prior to CFT073 stimulation for 4 or 6 h at MOI 1. Supernatants and total RNA were collected and kept at −80 °C until further analysis.
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