Phorbol 12 myristate 13 acetate pma

Doxorubicin hydrochloride was from ThermoFisher Scientific™ (Cat. No: J64000.MA, Waltham, MA, USA). SAC was from MedChemexpress Co., Ltd. (Cat. No. HY-N0319, Monmouth Junction, NJ, USA). Piceatannol was from Millipore (Burlington, MA, USA). Go 6983 and m-3M3FBS were purchased from Selleck Chemicals, LLC (Cat. No. S0982, Houston, TX, USA). Phorbol 12-myristate 13-acetate (PMA) was from Millipore. Anti-phosphotyrosine antibody, clone 4G10, was obtained from Millipore (Cat. No. 05-321X). Phospho-(Ser/Thr) Phe Antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). All antibodies for flow cytometry were obtained from BioLegend (San Diego, CA, USA). Alexa Fluor™ 647-conjugated Fibrinogen from human plasma was purchased from Sigma-Aldrich (Cat. No. F35200, St. Louis, MO, USA). Fibrinogen from human plasma was purchased from Sigma-Aldrich (Cat. No. F3879). Anti-GPIbβ antibodies conjugated with DyLight 649 were from Emfret Analytics (Eibelstadt, Bayern, Germany). CellTracker™ Green CMFDA Dye was from ThermoFisher Scientific™.

RPMI-1640 culture medium, fetal bovine serum bovine (FBS), Dulbecco's phosphate buffered saline (PBS), and Dextran 500 were purchased from Gibco™, Thermo Fisher Scientific Inc. (Waltham, MA, USA). Histopaque-1077, trypan blue, levamisole hydrochloride (C11H12N2S-HCl) and hyoscine butylbromide (catalogue number, H1450000), phorbol 12-myristate 13-acetate (PMA), and dihydrorhodamine 123 (DRH123) were obtained from Millipore Sigma (Burlington, MA, USA). The Cytometric Bead Array (CBA) Human Inflammatory Cytokine kit, and the anti-CD45-PeCy7 mAb (clone 30-F11) were purchased from Becton Dickinson (BD) Biosciences (San Diego, CA, USA). 3,3′-Dihexyloxacarbocyanine iodide (DIOC₆) and propidium iodide (PI) were bought from Invitrogen™ Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sytox Green and Hoechst 33342 were acquired from Thermo Fisher Scientific. The anti-MPO-PE (clone 2C7) and antineutrophil elastase (NE)-Alexa fluor 647 (clone 950317) mAbs were purchased from Novus Biologicals Biotechne (Littleton, CO, USA). Cocaine.HCI (1.0 mg base/1 mL methanol) (analytical reference standard COC-156-HC-1LM) was purchased from Lipomed AG (Arlesheim, Basel-Country, Switzerland) and imported via Químicos® complying with all permissions of the Fondo Nacional Antidrogas (FONA) of Colombia.

THP-1 cells, obtained from ATCC (TIB-202TM), were cultured at 37°C with 5% CO₂ in RPMI-1640 media (Gibco) containing 10% heat-inactivated fetal calf serum (Gibco), 10 mM HEPES (MilliporeSigma), 100 units/mL penicillin-streptomycin (MilliporeSigma), and 2 mM L-glutamine (Gibco). For SILAC, THP-1 cells were grown in L-glutamine, L-arginine, and L-lysine deficient RPMI-1640 (Caisson Labs), supplemented with 100 units/mL penicillin-streptomycin (MilliporeSigma), 2 mM L-glutamine (MilliporeSigma), 7% dialyzed fetal bovine serum (Gibco) and labeled with either L-lysine and L-arginine (Lys0, Arg0), L-lysine-²H₄ and L-arginine-¹³C₆ (Lys4, Arg6), or L-lysine ¹³C₆-¹⁵N₂ and L-arginine ¹³C₆-¹⁵N₄ (Lys8, Arg10). Stable isotope-labeled amino acids were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). For experiments, THP-1 cells were differentiated with 10 ng/mL of phorbol 12-myristate 13-acetate (PMA) (MilliporeSigma) for 16–18 h. The differentiated THP-1 cells (dTHP-1) were rested in fresh media for 6 h prior to treatments.

ABK5-1 was purchased from ChemBridge Corporation (San Diego, CA, USA) and ABK5-2 was from Enamine (Monmouth Junction, NJ, USA). [³H]CP,55940 and [³⁵S]GTPγS were purchased from PerkinElmer life Sciences (Waltham, MA, USA). CLCX12 was obtained from R&D Systems (Minneapolis, MN, USA). Phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) were purchased from Millipore Sigma (St. Louis, MO, USA).

THP-1 cells (TIB-202TM) were from ATCC. RPMI 1640 media and Fetal bovine serum (FBS) were purchased from Gibco. HEPES, penicillin/streptomycin, L-glutamine, Hanks’ balanced salt solution, M199 medium, folic acid, hemin, adenosine, Pepstatin A, Phorbol 12-myristate 13-acetate (PMA), PreScission enzyme (GE27-0843-01), and Amicon® Ultra-2 centrifugal filter devices (UFC200324) were purchased from Millipore Sigma. Anti-Ago1 (5053), anti-Ago2 (2897), anti-Ago3 (5054), anti-Lamin A/C (2032), anti-Calreticulin (12238), and anti-Calnexin (2679) were purchased from Cell Signaling. Anti-GST (SC-138), anti-HSC70 (HSP70) (SC-7298), and anti-Actin (SC-47778) were from Santa Cruz biotechnology. Anti-GAPDH (G041) was from Abm, and anti TRBP (Ab42018) was purchased from Abcam. RPMI 1640 media lacking L-glutamine, L-arginine, and L-lysine was purchased from Caisson labs. North2South Hybridization buffer (37549), North2South Hybridization Stringency Wash Buffer (37555), and Chemiluminescent Nucleic Acid Detection Module kit (89880) were purchased from ThermoScientific.