Anti syk

Cells were incubated at 37°C, 5% CO₂ for 5 min or 10 min for FcγR or integrin stimulation respectively. In some cases, BM neutrophils were infected with YPIII WT-Yptb or ΔyopH for 30 min and then spun down on the ligand coated surface. Cells were lysed in 1X Novex buffer and 2x10⁶ cell equivalents were resolved on a 4–12% NuPAGE gel (Invitrogen; Cat# NP0335BOX) in MOPS buffer. Proteins were transferred to Immobilon-FL filters and subjected to Western blot analysis with the following primary antibodies at a dilution of 1:500 anti-Syk (Cell Signaling; Cat# 2712), anti-SykpY352 (Cell Signaling Cat#2701), anti-SLP76 clone AS55 (Millipore; Cat# 05–1426), anti- SLP76pY128 (BD Pharmingen; Cat# 558367), anti-PLCγ2 (Cell Signaling; Cat# 3872), anti-PLCγ2pY1217 (Cell Signaling; Cat# 3871), anti-Erk1/2 pThr202/pTyr204 (Cell Signaling; Cat# 4370), anti-Erk1/2 (Cell Signaling; Cat# 9102), or at 1:1000 of anti-Rho-GDI (Cell Signaling; Cat# 2564S). Secondary LI-COR goat anti-mouse antibody IRDye 800 CW (Cat# 827–08364) and goat anti-rabbit IRDye 800 CW (Cat# 926–32211) were used at a dilution 1:20,000. ODYSSEY CLx LI-COR system was used to image the blots and IS Image Studio used for analysis and quantification of the bands.

Total proteins from the ischemic cortical area brain tissues or BV2 microglial cells were collected in equal amounts of cell lysate, and the separated proteins in the supernatant were subsequently transferred. For immunoblotting, the following primary antibodies were used: Anti-Dectin-1 (1:1,000; Abcam), anti-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-p-Syk (1:1,000; Cell Signaling Technology, Inc.), anti-TNF-α (1:1,000; Cell Signaling Technology, Inc.), and anti-iNOS (1:1,000; Cell Signaling Technology, Inc.). Western blotting was performed at 6 h and 3, 5, and 7 days after ischemic stroke for the mouse tissues and at 0, 3, 6, and 12 h after OGD/R treatment and 3, 6, 12, and 24 h after LPS treatment for the BV2 microglial cells. Then, the optimal time points of 3 days for the in vivo experiments and 3 h for OGD/R treatment and 24 h for LPS treatment in vitro were selected for the subsequent experiments.

GM‐CSF and M‐CSF/IL‐4 BMMs were differentiated over 8 days and stimulated with 40 or 100 µg/mL TDB for 6 h. Whole cell lysate was collected with NP‐40 buffer containing protease (cOmplete Tablets, Roche, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche). Western blots were performed by 10% SDS–PAGE and wet blotting. Antibodies used were anti‐P‐Syk(Y519/520), anti‐P‐Syk(Y317), anti‐Syk, anti‐β‐Actin, anti‐rabbit IgG‐HRP (Cell Signaling Technology, MA, USA) and anti‐P‐SHP2 (Y542), anti‐P‐SHP2(Y580) and anti‐SHP2 (Invitrogen, CA, USA). Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, IL, USA) and imaged with Amersham Imager 600.

Cells or periostea were lysed in 1X RIPA (Millipore, MA, USA). A BCA Protein Assay Reagent Kit (Pierce, MO, USA) was used to measure protein concentrations. Total protein was transferred to nitrocellulose membranes (Bio‐Rad) and incubated overnight with primary antibody: rabbit polyclonal anti‐Syk, anti‐P‐Syk (phospho‐Y352), anti‐c‐Fos, anti‐Erk1/2, anti‐P‐Erk, anti‐NFATc1, anti‐CD11b and anti‐β‐actin (Cell Signaling Technology, Boston, USA). Blots were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (donkey anti‐rabbit IgG, Jackson Immunoresearch, Baltimore, USA) in blocking buffer. Quantification of the immunoblots was performed using Image J software (version 1.49n, NIH Image) after exposed to X‐ray film, and results were normalized to β‐actin 7.

UTP, ATP, ADP, PGE₁, PGE₂, 2,4-dinitrophenyl human serum albumin (DNP-HSA), anti-DNP IgE (clone SPE-7), p-nitrophenyl N-acetyl-β-d-glucosaminide, and the GenElute Mammalian Total RNA miniprep kit were obtained from Sigma-Aldrich (Tokyo, Japan). Allophycocyanin (APC)-conjugated rat anti-mouse c-Kit antibodies (clone 2B8) were obtained from BD Pharmingen (Tokyo, Japan). Phycoerythrin (PE)-conjugated mouse anti-mouse FcεRIα antibodies (clone MAR-1) were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse interleukin (IL)-3 and recombinant mouse SCF were obtained from Peprotech (London, UK). MRS2179, AR-C118925, NF449, AZ10606120, 5-BDBD, Ivermectin, PP2, LY303511, LY294002, Triciribine, and AS605240 were obtained from Tocris Bioscience (Bristol, UK). R406 was obtained from Cayman Chemical (Michigan, USA). Fura-2-acetoxymethylester (AM) and PTX were obtained from Wako (Osaka, Japan). Anti-phospho-Syk, anti-Syk, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). ONO-DI-004 (selective EP1 agonist), ONO-AE1-259-01 (selective EP2 agonist), ONO-AE-248 (selective EP3 agonist), and ONO-AE1-329 (selective EP4 agonist) were obtained from ONO Pharmaceuticals (Osaka, Japan). All other chemicals were of reagent-grade or the highest quality available.