Syk d3z1e

All of the chemical reagents were obtained from Sigma-Aldrich, unless otherwise stated. Abs used in the flow cytometry were obtained from Biolegend, unless otherwise indicated, and include those against mouse CD80 (GL-1), I-A^b (AF6–120.1), human Dectin-2 (R&D systems, 545943), CD3 (OKT3), CD14 (M5E2), CD16 (3G8), CD19 (HIB19), CD20 (2H7), CD56 (HCD56), CD11c (3.9), and HLA-DR (L243). The Abs used for Western blot were obtained from Cell Signaling and include Syk (D3Z1E), phospho-Syk (C87C1), p38 (D13E1), phospho-p38 (D3F9), IkB (rabbit polyclonal Ab), and β-actin (13E5). LPS from H. alvei PCM 1223, S. enterica O66, K. pneumoniae O1, E. coli O9a, and the rough mutant were isolated as described previously (28 (link), 42 (link), 45 (link), 61 (link), 62 (link)). The lipid A from the Man-LPS was isolated by hydrolysis as described previously (28 (link)). Galactan and β-linked mannans were from Megazyme. The Syk inhibitor R406 was purchased from InvivoGen. ELISA kits for mouse and human TNFα and IL-10 were from Biolegend and were used according to the manufacturer's instructions.

Splenic B cells were purified by negative selection of CD43⁺ cells using anti-CD43 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purified B cells (1 × 10⁷ cells/ml) were stimulated with 10 µg/mL anti-mouse IgM F(ab)′2 (Jackson ImmunoResearch, West Grove, PA, USA) or 10 µg/mL anti-human IgM F(ab)′2 (Jackson ImmunoResearch) and then lysed in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 and 0.5 mM EDTA plus protease and phosphatase inhibitor cocktails (Nacalai Tesque)]. Samples were transferred to polyvinylidene difluoride membranes by electrophoresis and analysed by immunoblotting with antibodies against p-Syk (Tyr525/526) (C87C1) and Syk (D3Z1E) (Cell Signaling Technology, Danvers, MA, USA). The purified splenic B cells were cultured with or without 25 ng/ml BAFF (R&D Systems, Minneapolis, MN, USA) for 24, 48, or 72 h. The frequency of live B cells was assessed using TOPRO3 (Invitrogen) exclusion.

To validate the fold increase protein expression and phosphorylation changes as observed in the antibody array for OA triggered T98G cells, all individual OA and NP-CSF triggered T98G cell lysates (n = 15 each) were checked for protein expression level of targets with western blotting. Ten micrograms of each CSF-triggered T98G cell lysates were loaded per lane of 10% Tris-glycine gels and run using Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on gel were transferred onto Polyvinylidene difluoride (PVDF) membrane. 0.1% Tween-20 added into Tris-buffered saline (TBS-T) was used to wash membranes and prepare antibodies. Non-specific binding sites on the membranes were blocked in 5% w/v Bovine serum albumin (BSA)-TBS-T or milk-TBS-T. BSA was used when checking for spleen tyrosine kinase (Syk); while milk was used for STE20-related kinase adaptor protein (STRAD) and GAPDH. Primary antibodies used in this study are: STRAD (G-8) (Santa Cruz Biotechnology, CA, USA); Syk (D3Z1E) (Cell Signaling Technology Inc., MA, USA); and GAPDH (MAB374) (EMD Millipore, Darmstadt, Germany). GAPDH, with a molecular weight of 37 kDa, was used as the loading control for normalization. Membranes were imaged with ChemiDoc XRS+ system. Densitometry quantification was done with ImageLab software.