LA-4 cells were pre-incubated with the indicated amounts of either the mTOR inhibitor rapamycin (Invivogen, Toulouse, France), MAPK-inhibitors U0126 (MEK1/2 MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands), SP600125 (SAP/JNK MAPK inhibitor, Invivogen), SB-202190 (p38α/β MAPK inhibitor, Invivogen), the NFκB- and MAPK-inhibitor dexamethasone (Invivogen), the IKK-β-inhibitor TPCA-1 (Abcam, Berlin, Germany), the inhibitor of actin polymerization cytochalasin A (Sigma-Aldrich), the inhibitor of endosomal acidification chloroquine (InvivoGen), or the COX2-inhibitor NS-398 (Sigma-Aldrich, Steinheim, Germany) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. For analyzing cell viability, LA-4 cells were treated as indicated and stained for dead cells using the fixable viability dye eFlour450 (#65-0865-14, eBioscience) and measured by FACS. Data were analyzed using FlowJo V.7 (Treestar Inc., Ashland, OR, USA) and GraphPad PRISM (GraphPad Software, San Diego, CA, USA).
Tpca 1
Manufactured by Abcam
Sourced in United Kingdom
TPCA-1 is a chemical compound used in research applications. It functions as an inhibitor of the IKK-beta enzyme, which is involved in the activation of the NF-kappa-B transcription factor pathway.
Automatically generated - may contain errors
Lab products found in correlation
Sb202190, by InvivoGen (3 mentions)
Sp600125, by InvivoGen (3 mentions)
U0126, by Cell Signaling Technology (3 mentions)
Bay 11 7082, by Abcam (3 mentions)
Eflour780, by Thermo Fisher Scientific (2 mentions)
Bms 345541, by Abcam (2 mentions)
Cytochalasin a, by Merck Group (1 mentions)
Eflour450, by Thermo Fisher Scientific (1 mentions)
Ns 398, by Merck Group (1 mentions)
Chloroquine, by InvivoGen (1 mentions)
Rapamycin, by InvivoGen (1 mentions)
Dexamethasone (dex), by InvivoGen (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Human fc block, by BD (1 mentions)
Cytochalasin d, by Fujifilm (1 mentions)
Methyl β cyclodextrin, by Merck Group (1 mentions)
Pyrrolidine dithiocarbamate pdtc, by Abcam (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Mcc950, by InvivoGen (1 mentions)
Vx 765, by InvivoGen (1 mentions)
8 protocols using tpca 1
1
Inhibition of Immune Signaling Pathways
2
Inhibiting Immune Response Pathways
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For inhibition experiments mDCs were pre-incubated with the indicated amounts of rapamycin (mTOR inhibitor), BAY-11-7082 (irreversible inhibitor of TNF-α-induced IkB-α phosphorylation resulting in inactivation of NFkB), triptolide (NFκB inhibitor), dexamethason (NFκB and MAPK inhibitor), the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) and BMS-345541 (Abcam, Cambridge, UK), as well as the MAPK inhibitors SP600125 (JNK MAPK inhibitor, Invivogen, Toulouse, France), SB-202190 (p38α/β MAPK inhibitor, Invivogen, Toulouse, France), or U0126 (MEK1/2 MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with equimolar amounts of rFlaA + rBet v 1 or rFlaA:Betv1 for either 30 min (Western Blot), 24 h (ELISA and cytotoxicity), or 72 h (ELISA and analysis of cell metabolic state). Toxicity of the used inhibitors was determined using the fixable viability dye eFlour780 (Thermo Fisher Scientific, Darmstadt, Germany, Repository Figure S1 ). Inhibitor concentrations showing toxic effects were excluded from the analysis.
3
Fc Fragment Modulation Techniques
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Human IgG F(ab’)2 fragments (Gamma Venin P) were purchased from Sanofi, Paris, France. N-acetyl cysteine (NAC) (Sigma, St Louis, MO), pyrrolidine dithiocarbamate (PDTC) (Abcam), TPCA-1 (Abcam), methyl-β-cyclodextrin (Sigma) and cytochalasin D (Wako, Osaka, Japan) were also purchased. Human BD Fc Block was purchased from BD Biosciences, Franklin Lakes, NJ.
4
Inhibition of Inflammatory Pathways in THP-1 Macrophages
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PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 (InvivoGen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 (InvivoGen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, InvivoGen), SB202190 (p38α/β MAPK inhibitor, InvivoGen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in Supplementary Figure 2
5
Splenic NK Cell Cytokine Stimulation
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Freshly sorted primary splenic NK cells were expanded, then stimulated with indicated cytokines for 2-24hrs according to the experiment: IL-12 (R&D, 1 ng/mL), IL-18 (R&D, 10 ng/mL), and a combination of both. After stimulation, cells were subjected to RNA isolation, chromatin isolation, and protein extraction, and cultured supernatant was collected and subjected to ELISA. For NF-kB inhibition, cells were treated with IL-12/IL-18 and TPCA-1 (Abcam, 5 mg/mL) for 4 hours. Cells were subjected to RNA isolation and protein purification, and cultured supernatant was collected and subjected to ELISA.
6
TPCA1 and Tunicamycin Induced ER Stress Response
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SW1353 cells with 50% confluence were treated with 10 μM TPCA1 (Abcam, Cat. no. ab145522), 2 μM tunicamycin (SIGMA, Cat. no. T7765), 10 μM TPCA1 + 2 μM tunicamycin, or DMSO (SIGMA, Cat. no. D2650-100ML) in a chamber slide for 48 h. Then, cells were fixed with 4% PFA (SIGMA, Cat. no. 03112DH) for 15 min at room temperature followed by permeabilization with 0.5% Triton-x100 (SIGMA, Cat. no. SLBH9920V) for 5 min at room temperature. Next, blocking was performed at room temperature using 10% normal goat serum (Thermo, Cat. no. 50062Z) for 2 h, after which cells were incubated with the primary antibody CHOP (Abcam, Cat. no. ab11419; dilution, 1:100) at 4 °C overnight. Then, cells were incubated with fluorescence-conjugated secondary antibody Alexa Fluor® 488 (Abcam, Cat. no. ab150117; dilution. 1:1000) at room temperature for 1 h. Finally, the slides were mounted using a mounting medium with DAPI (VECTASHIELD, Cat. no. H-1200). Fluorescent images were obtained using a Carl Zeiss LSM980 laser scanning confocal microscope (Carl Zeiss, Germany) with a 20x objective.
7
Investigating NF-κB Inhibitors in Oxaliplatin and Curcumin Treatment
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Oxaliplatin and curcumin were purchased from FUJIFILM Wako Pure Chemical. Three known NF‐κB inhibitors (Bay11‐7082; IκBα kinase inhibitor, TPCA1; IκB kinase inhibitor, MG132; proteasome inhibitor) were purchased from Abcam for Bay11‐7082 and TPCA1, or from Cayman Chemical for MG132, respectively. CMG was synthesized using a previous method (KNC Laboratories).21
8
Recombinant Proteins and Inhibitor Reagents for Cell Signaling
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Recombinant proteins were purchased from R&D Systems: EDA-A1 (3944-ED), EDA-A2 (922-ED), TNFα (410-MT), BAFF (8876-BF), TWEAK (1237-TW), OSM (495-MO), IL-6 (406-ML) and LIF (8878-LF). Small molecule inhibitors were acquired from indicated sources: TPCA-1 (abcam, ab145522), MG132 (Sigma; M8699), BAY 11-7082 (abcam, ab141228), BOT-64 (Santa Cruz; sc-222062), B022 (Aobious; AOB8699). Mouse EDA-A1 (MC208411), mouse EDA-A2 (MC208415) and mouse OSM (MR226014) expression plasmids were purchased from Origene.
Mouse NIK expression plasmid was purchased from Invivogen (pUNO1-mMap3k14). Wild-type human NIK and the NIK-K429/430A mutant plasmids were a gift from Prof. Michael Kracht (JLU Giessen).
Mouse NIK expression plasmid was purchased from Invivogen (pUNO1-mMap3k14). Wild-type human NIK and the NIK-K429/430A mutant plasmids were a gift from Prof. Michael Kracht (JLU Giessen).
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