Anti syk

Platelet samples for Western blot analysis were prepared by adding 3× Lämmli buffer (200 mM tris/HCl, 15% (v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromphenol blue, 1:10 β-mercaptoethanol) directly in the aggregation cuvettes to stop platelet responses, then boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using 8% SDS-polyacrylamide gels followed by immunoblotting as previously described [28 (link)].
Phospho-antibodies against Syk S297, Syk Y525/526, Syk Y352, LAT Y191, PLCγ2 Y759, and MARCKS S159/163 (Cell Signaling Technologies, Danvers, MA, USA) were used diluted 1:1000 in 5% BSA or 1:700 for MARCKS. Blots probed with phospho-antibodies were stripped and reprobed with a corresponding antibody detecting the total protein, anti-Syk, or anti-PLCγ2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or other loading controls anti-β-actin or anti-α-actinin (Cell Signaling Technologies, Danvers, MA, USA) diluted 1:1000 in 5% BSA. After incubation with the appropriate secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (BioRad Laboratories, Hercules, CA, USA) enhanced chemiluminescence (ECL) detection was performed using Fusion FX7 (Vilber Loumat GmbH, Eberhardzell, Germany).

Anti-p-p65 antibody, Gö6976, heparin, PMA, Röttlerin, and PKC-δ siRNA were purchased from Sigma-Aldrich (St.Louis, MO). Bay 11-7082, Bay 61-3606, piceatannol, and caffeic acid phenethyl ester (CAPE) were purchased from Cayman Chemicals (Ann Arbor, MI). Lipofectamine 3000 reagent was acquired from Invitrogen Life Technologies (Gaithersburg, MD). Antibody specific for Thy-1, and p-PKC-δ was purchased from Cell Signaling Technology (Danvers, MA). Anti-Syk, p65, PKC-δ, and G3PDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for p-Syk was purchased from EMD Millipore (Temecula, CA). Dual-Luciferase assay kit was purchased from Promega (Madison, WI). Penicillin and streptomycin were purchased from HyClone (South Logan, Utah). Endothelium cell growth factor (ECGS) was purchased from Biomedical Technologies (Stoughton, MA). Fetal calf serum (FCS), Medium 199 (M199), and GlutaMAX Supplement were obtained from GIBCO (Grand Island, NY). Endothelial Cell Growth Medium MV was purchased from PromoCell (Heidelberg, Germany).

For the analysis of Erk phosphorylation, DT40 Syk-AQL-EGFP cells were treated with or without goat-anti-mouse IgM (10 (μg/mL) for the indicated periods of time at 37 °C and then lysed in buffer containing 25% sucrose, 2.5% SDS, 25 mM Tris/2.5 mM EDTA, 2.5 mg pyronin Y, and 2% 2-mercaptoethanol. The DNA in lysates was sheared by passing through a 26 G × 1/2 in needle. Proteins in the lysate were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed by Western blotting with anti-pERK (Cell Signaling p44/p42 MAPK (T202/Y204) rabbit 4370S), and anti-Syk (Santa Cruz N-19 rabbit) antibodies. The results of these assays were used for parameter screening and will be referenced in the results section.

Sanguinarine (13-methyl(1,3) benzodioxolo (5,6-c) -1,3-dioxolo(4,5-i) phenanthridinium), (HPLC ≥ 98%, SA, Figure 1) (Absin, Shanghai, China), was dissolved in 0.1% DMSO (final concentration). The EDTA, prostaglandin E1(PGE1) and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The CHRONO-LUME reagent and collagen were acquired from Chrono-Log Corp. (Havertown, PA, USA). The collagen-related peptide was obtained from Dr. Newman’s lab (Blood Center of Wisconsin, Milwaukee, WI, USA). The FITC-conjugated anti-CD62P (P-selectin) antibody and FITC-conjugated anti-PAC-1(αIIbβ3) antibody were purchased from Biolegend (San Diego, CA, USA). The Fluo-3AM was purchased from MCE (Shanghai, China). The CellTrace Calcein Green was purchased from Invitrogen (Carlsbad, CA, USA). The anti-phospho-PI3K(Ser1070), anti-PI3K, anti-phospho-Akt(Ser473), anti-phospho-GSK3β (Ser9), anti-phoshpo-PLCγ2 (Tyr1217), anti-β3, anti-phospho-β3 (Tyr474), anti-Src, anti-phospho-Src (Tyr416) and anti-phospho-Syk (Tyr525/526) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-Syk, anti-PLCγ2, anti-Akt and antil-GSK3β were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

All reagents were purchased from Thermo Fisher Scientific unless otherwise stated. Chrono-lume, used for the detection of secreted ATP, was purchased from Chrono-log corporation. Anti-pSyk Y525/526 (mouse Y519/520) and anti-pPLCγ2 Y1217 were purchased from Cell Signaling Technology. Anti-pSyk Y352 (Y346 in mice) and anti-pSyk Y348 (Y342 in mice) were purchased from Abcam. Anti-Syk and anti-PLCγ2 were purchased from Santa Cruz Biotechnology. Ibrutinib was purchased from Selleckchem. Odyssey blocking buffer and secondary antibodies IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse were purchased from Li-Cor. CRP-XL was purchased from Dr Richard Farndale at the University of Cambridge. AYPGKF was purchased from GenScript.

Thrombin, ADP, and U46619 were ordered from Sigma (St. Louis, MO, USA). Collagen and CHRONO-LUME reagent were purchased from Corp (Havertown, PA, USA). IV.3 Fab was kindly provided by Prof. Peter Newman (Blood Center, Wisconsin, USA). Anti-syk, anti-PLCγ2, and RP215-HRP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Anti-FcγRIIa antibody was obtained from Abcam (Massachusetts, US). Anti-phospho-PLCγ2 (Tyr1217) and anti-phospho-syk (Tyr525/526) antibodies were obtained from Cell Signaling (Beverly, MA, USA).