All drugs and chemicals used in the following experiments were purchased from Sigma (St Louis, MO) unless stated otherwise. The PLC inhibitor U73122, the TRPV1 antagonist SB366791, the PKC inhibitor GF‐109203 X, and the inactive analogue of the PKC inhibitor were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). The PIP2 analogue PI(4,5)P2‐diC8 was purchased from Echelon Biosciences Inc. (Salt Lake City, UT).
U73122
Manufactured by Enzo Life Sciences
Sourced in United States
U73122 is a chemical compound that functions as a selective, cell-permeable phospholipase C inhibitor. It acts by interfering with the activity of phospholipase C enzymes, which play a role in cellular signal transduction processes.
Automatically generated - may contain errors
Lab products found in correlation
Gf109203x, by Enzo Life Sciences (1 mentions)
Pi 4 5 p2 dic8, by Echelon Biosciences (1 mentions)
Rottlerin, by Enzo Life Sciences (1 mentions)
Reparixin, by MedChemExpress (1 mentions)
Venetoclax, by Chemietek (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Anti calnexin, by Enzo Life Sciences (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Biotium (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Dantrolene, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Ly294002 hydrochloride, by Merck Group (1 mentions)
Z yvad fmk, by Abcam (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Z devd fmk, by Abcam (1 mentions)
4 pba, by Merck Group (1 mentions)
5 protocols using u73122
1
Pharmacological Modulation of TRPV1
2
Inhibition of Inflammatory Signaling
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PLAG and PLH were obtained from Enzychem Lifesciences Corporation (Daejeon, South Korea). Gemcitabine hydrochloride was purchased from Dong-A ST (Seoul, South Korea). Reparixin was purchased from MedChem Express (NJ, USA). N-acetyl-l-cycteine, diphenyleneiodonium were purchased from Sigma-Aldrich (MO, USA). U73122 and Rottlerin were purchased from ENZO Life Sciences (NY, USA).
3
Investigating IP3 Signaling and Apoptosis
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Reagents were as follows: ethylene glycol tetraacetic acid (EGTA) (Acros Organics, Geel, Belgium, 409910250), Fura-2 AM (Biotium, Kampenhout, Belgium, 50033), Annexin V-Fluorescein isothiocyanate (FITC) (Becton Dickinson, Franklin Lakes, NJ, USA, 556419), 7-aminoactinomycin D (7-AAD) (Becton Dickinson, 555815), U73122 (Enzo Life Sciences, Farmingdale, NY, USA, BML-ST391-0005), U73343 (Enzo Life Sciences, BML-ST392-0005), venetoclax (ChemieTek, Indianapolis, IN, USA, CT-A199), anti-human IgG/M (Jackson ImmunoResearch, West Grove, PA, USA, 109-006-127). The following antibodies were used: anti-IP3R2 (Abiocode, Agoura Hills, CA, USA, R2872-3); anti-calnexin (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SPA-865-D); anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc7382HRP); anti-Bim (Bioké, Leiden, The Netherlands, 2819 S); anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA, G8795); anti-vinculin (Sigma-Aldrich, Munich, Germany, V9131). The sequences of the peptides used in this study were: BIRD-2 (RKKRRQRRRGGNVYTEIKCNSLLPLAAIVRV) and TAT-Ctrl (RKKRRQRRRGGSIELDDPRPR). These peptides were synthesized by LifeTein (South Plainfield, New Jersey, USA) with a purity of at least 85%. The IP3 sponge (pEF-GSTm49-IRES-GFP) is a protein constructed from the IP3-binding core of the type 1 IP3R with a single amino acid substitution (R441Q) that has a very high affinity for IP3 [29 (link)].
4
Mechanistic Insights into A. hydrophila Infection
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C. gariepinus and A. hydrophila (Strain 500,297) were used in the study. The protocols for HKM isolation, and infection of HKM with A. hydrophila (MOI 1:50) have been described earlier [14 (link)].
zHKMs were incubated separately with ER stress inhibitor (4-PBA, 10 µM, Sigma), mtROS inhibitor (YCG063, 10 µM, Calbiochem), ETC inhibitor (antimycin A, 50 µM, Sigma), MPTP inhibitor (Cyclosporin A, 5 µM, Sigma), RyR inhibitor (Dantrolene, 10 µM, Sigma), Cytochalasin D (Cyt D, 5 µg/mL, Sigma), Autophagy inducer (Rapamycin, 20 µM, Sigma), Thapsigargin (1 µM, Sigma), Akt inhibitor (124,005, 10 µM, Calbiochem) for 1 h and mitochondrial Ca2+ uptake inhibitor (Ru360, 10 µM, Calbiochem), Caspase-1 inhibitor (Z-YVAD-FMK, 7.5 µM, Biovision), Caspase-3 inhibitor (Z-DEVD-FMK, 10 µM, Biovision), IP3R inhibitor (2-APB, 100 μM, Sigma), PLC inhibitor (U73122, 2 μM, Enzo Life Science), PI3-Kinase inhibitor (LY-294002 hydrochloride, 14.5 μM, Sigma), intracellular Ca2+chelator, (BAPTA-AM, 5 mM, Sigma), and JNK inhibitor (SP600125, 10 µM, Calbiochem) for 2 h followed by A. hydrophila infection as mentioned earlier [3 ]. The inhibitor concentrations had no adverse effects on HKM viability and bacterial growth (data not shown).
zHKMs were incubated separately with ER stress inhibitor (4-PBA, 10 µM, Sigma), mtROS inhibitor (YCG063, 10 µM, Calbiochem), ETC inhibitor (antimycin A, 50 µM, Sigma), MPTP inhibitor (Cyclosporin A, 5 µM, Sigma), RyR inhibitor (Dantrolene, 10 µM, Sigma), Cytochalasin D (Cyt D, 5 µg/mL, Sigma), Autophagy inducer (Rapamycin, 20 µM, Sigma), Thapsigargin (1 µM, Sigma), Akt inhibitor (124,005, 10 µM, Calbiochem) for 1 h and mitochondrial Ca2+ uptake inhibitor (Ru360, 10 µM, Calbiochem), Caspase-1 inhibitor (Z-YVAD-FMK, 7.5 µM, Biovision), Caspase-3 inhibitor (Z-DEVD-FMK, 10 µM, Biovision), IP3R inhibitor (2-APB, 100 μM, Sigma), PLC inhibitor (U73122, 2 μM, Enzo Life Science), PI3-Kinase inhibitor (LY-294002 hydrochloride, 14.5 μM, Sigma), intracellular Ca2+chelator, (BAPTA-AM, 5 mM, Sigma), and JNK inhibitor (SP600125, 10 µM, Calbiochem) for 2 h followed by A. hydrophila infection as mentioned earlier [3 ]. The inhibitor concentrations had no adverse effects on HKM viability and bacterial growth (data not shown).
5
Signaling Pathway Antibodies and Inhibitors
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Antibodies directed against P-PKB (Ser473; #4051), P-ERK1/2 (Thr202/Tyr204, #4370), P-IKKα/β (Ser176/Ser177, #2078), P-IκBα (Ser32, #2859), P-LAT (Tyr191, #3584), P-p38 (Thr180/Tyr182, #9216), P-PLCγ2 (Tyr759, #3874), and PI3K p85 (#4292) were purchased at Cell Signaling Technologies. Anti-GAPDH (sc-32233), anti-BTK (sc-1696), and anti-P-PLCγ1 (Tyr783; sc-12943) antibodies were from Santa Cruz Biotechnology and anti-PLCγ1 (#2112-1) antibody wase obtained from Epitomics. The antibody against P-BTK (Tyr551; #MAB7659) was purchased at R&D Systems. The following inhibitors were used: for BTK, Ibrutinib (PCI-32765, A11020, Adooq Bioscience), for PLC, U73122 (BML-ST391, Enzo) as well as its inactive compound U73343 (BML-ST392, Enzo), and for PI3K, Wortmannin (#681676, Merck Millipore). Dinitrophenyl (DNP)-human serum albumin (HSA) containing 30–40 mol DNP per mol albumin (A6661) and anti-DNP-specific monoclonal IgE (clone SPE-7, D8407) were purchased at Sigma-Aldrich. DMSO (A3672) was obtained from PanReac Applichem and Phorbol-12-myristate-13-acetate (PMA, #524400) from Merck Millipore.
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