All activators and inhibitors were of analytical grade and freshly prepared from frozen stock solution. The stock solution of LPA was 5 mM dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA). Pertussis toxin (PTX, Sigma-Aldrich) was dissolved at a concentration of 250 µg/mL in H2O. U0126 as well as Wortmannin (both Sigma-Aldrich) were dissolved at 10 mM in DMSO (Roth, Karlsruhe, Germany). Gö6976 (Tocris Biosciences, Bristol, UK) was dissolved at 1 mM in DMSO and ω-agatoxin-TK (Peptanova, Sandhausen, Germany) at 100 µM in H2O. For AlF4−, 30 µM AlCl3 and 10 mM NaF were mixed. FK506 (InvivoGen, Toulouse, France) was dissolved at 20 mM in DMSO and Cyclosporin A (Alfa Aesar, Ward Hill, MA, USA) at 1 mM in ethanol.
G 6976
Manufactured by Bio-Techne
Sourced in United Kingdom
Gö6976 is a selective inhibitor of protein kinase C (PKC) isoforms. It acts by binding to the catalytic domain of PKC, thereby preventing its activation and downstream signaling.
Automatically generated - may contain errors
Lab products found in correlation
G 6983, by Bio-Techne (4 mentions)
Gf109203x, by Bio-Techne (3 mentions)
Wortmannin, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Sf016 200, by Merck Group (2 mentions)
Cgp77675, by Bio-Techne (2 mentions)
Chir99021, by Bio-Techne (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Sotrastaurin, by Selleck Chemicals (2 mentions)
Tamoxifen citrate, by Bio-Techne (2 mentions)
Myriocin, by Cayman Chemical (2 mentions)
Mpp dihydrochloride, by Bio-Techne (2 mentions)
Pkcζ ps, by Bio-Techne (2 mentions)
Dl pdmp, by Bio-Techne (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fk506, by InvivoGen (1 mentions)
Cyclosporin a, by Thermo Fisher Scientific (1 mentions)
21 protocols using g 6976
1
Preparation of Bioactive Lipid Solutions
2
RAW 264.7 Macrophage Cell Culture and Reagents
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RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC TIB-71, Manassas, VA; Lots 70000171, 70012232 with cell identity confirmed by ATCC via growth properties, morphology, and COI assay). Cell media (supplemented DMEM) was composed of DMEM obtained from Thermo Fischer Scientific (Waltham, MA), fetal bovine serum (FBS, contains ~400 μM BSA as specified by manufacturer) from Millipore-Sigma (St. Louis, MO), penicillin, streptomycin and GlutaminePlus (L-alanyl-L-glutamine) from Atlanta Biologicals (Flowery Branch, GA). Dulbelcco's phosphate buffered saline (D-PBS) was from Gibco (Gaithersburg, MD). All cell media was regularly subjected to mycoplasma testing to ensure lack of infection. Matriplate 96 well imaging plates were from Matrical, Inc. (Spokane, WA). Acetylsalicylic acid (Lot SLBV2290), ibuprofen (Lot SLBX6584), acetaminophen (Lot SLBR2060V), diclofenac (Lot BCBS5961V), wortmannin (Lot 086M4026V), PDGF (Lot SLB63548V), and HEPES (free acid) were obtained from Millipore-Sigma (St. Louis, MO). Gö6976 (Lot 6A/174381) was purchased from Tocris Biosciences (Minneapolis, MN). CellMask Deep Red and Green membrane stains were purchased from ThermoFischer Scientific (Waltham, MA). Cell-culture grade DMSO was purchased from MP Biomedicals (Santa Ana, CA). AKT-PH-mRFP mammalian expression plasmid was subcloned by John H. Evans [26 (link), 30 (link)].
3
Cell Culture and Signaling Inhibitors
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HUVECs were cultured in EBM2+ BulletKit (cells and media from Lonza, Rockville, MD). CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in high-glucose DMEM supplemented with 10% fetal bovine serum and antibiotic–antimycotic solution (all from Life Technologies/ThermoFisher Scientific, Grand Island, NY). Y-27632 (ROCK inhibitor) was purchased from Millipore (Billerica, MA). Cell-permeable C3 (RhoA inhibitor) transferase was purchased from Cytoskeleton (Denver, CO). U0126 (MEK inhibitor), LY294002 (PI3K inhibitor), Su6656 (Src family kinase inhibitor), AG 490 (JAK2 inhibitor), Gö6976 (PKCα inhibitor), FAK inhibitor 14, and PKCζ pseudosubstrate were from Tocris (Minneapolis, MN). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
4
A549 Cell Culture and PKC Inhibitor Assay
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Authenticated human A549 lung adenocarcinoma cells were obtained from ATCC (Manassas, VA) and cultured in RPMI medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin). PMA was purchased from Sigma-Aldrich (St. Louis, MO) PKC inhibitors GF109203X and Gö6976 were purchased from TOCRIS (Bristol, UK).
5
Visualizing Processed and Unprocessed vWF
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Rabbit anti‐vWF (cat. No. A0082) and rabbit anti‐vWF‐HRP, which stain both processed and unprocessed forms of vWF, were from DAKO (termed “total vWF”), two anti‐vWF antibodies against a neo‐epitope exposed at the carboxy terminus of the propeptide upon furin cleavage14 to visualize processed vWF (termed “pro‐vWF”), were used in this study. These "pro‐vWF" antibodies allowed visualization of only that vWF packaged into WPBs (thus excluding background staining of ER‐localized vWF). the first was a kind gift from Dr T Carter (St. George's University London), the second was produced by Eurogentec. Hoechst 33342 was from Life Technologies. Cytochalasin E (CCE) was purchased from Sigma, GÖ6976 was from TOCRIS. Histamine was from Enzo Life Sciences (cat. No. ALX‐550‐132); Phorbol 12‐Myristate 13‐Acetate (PMA), adrenaline, and 3‐isobutyl‐1‐metylxanthine (IBMX) were from Sigma; thrombin was from Calbiochem.
6
Adrenal H295R Cell Culture and Transfection
7
Cdx2-Inducible Mouse ES Cell Line
8
Osteoblast and Osteocyte Cell Culture
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Cell culture and experiments with UMR106 rat osteoblast-like cells (purchased from ATCC, Manassas, VA, USA) were conducted as described before [39 (link)]. Briefly, cells were cultured in DMEM high-glucose medium containing 10% FBS and penicillin-streptomycin at 37°C and 5% CO2.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
9
Antibody-Based Protein Trafficking Analysis
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Antibodies used were Mouse Anti-HA (Sigma, H9658), AP-3 (SA4, Developmental Studies Hybridoma Bank) and APP C-terminal (Sigma, A8717). The PKC activator Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma (P8139) and 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) was purchased from (Sigma, D5318). Staurosporine was purchased from Millipore (Cat No. 569397). Gö6976 was purchased from Tocris Bioscience (Cat. No. 2253).
10
Evaluation of Novel Signaling Modulators
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Tamoxifen citrate, ceramide, MPP dihydrochloride, G-1, Gö 6983, Gö 6976, PKCζ-PS and DL-PDMP were all obtained from Tocris Bioscience (Bristol, UK). Myriocin was obtained from Cayman Chemical (Ann Arbor, MI). Unless otherwise noted, all other chemicals were obtained from Sigma Aldrich.
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