Total syk

Immunoblotting has been described previously (1 (link)). Primary antibodies used include: SIRPα (1:1,000; #13379; Cell Signaling), phospho-Shp1 (1:1,000; Tyr564; #8849; Cell Signaling), total Shp1 (1:1,000; #3759; Cell Signaling), phospho-Shp2 (1:1,000; Tyr580; #3703; Cell Signaling), total Shp2 (1:1,000; #3397; Cell Signaling), phospho-Lck (1:1,000; Y394; #ab201567; Abcam), total Lck (1:1,000; #2752; Cell Signaling), phospho-SYK (1:1,000; Tyr525/526; #2710; Cell Signaling), total SYK (1:1,000; #13198; Cell Signaling), phospho-AKT (1:1,000; Ser473, #5082; Cell Signaling), total AKT (1:1,000; #9272; Cell Signaling), GAPDH (1:5,000; #5174; Cell Signaling), and β-actin (1:5,000; sc47778; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Cell Signaling) and goat anti-mouse secondary antibodies (1:5,000; Sigma-Aldrich) were used for detection. SignalFire™ Plus ECL reagent (#12630S, Cell Signaling) was used for protein expression detection as per the manufactures protocol. Fusion SL (Analis Instruments) was used as per manufacturer instructions and chemiluminescent captured using Fusion SL (Analis Instruments). Where protein expression has been quantitated, results represent relative protein levels normalized to corresponding total protein levels or β-actin or GAPDH using Image J software.

Primary BMDCs were obtained from 9-week-old C57BL/6 mice that were maintained under standard specific pathogen-free conditions. BMDCs were differentiated for 7 days in Roswell Park Memorial Institute medium (Gibco) and granulocyte-macrophage colony-stimulating factor. On day seven, BMDCs were seeded in 96-well plates at 10⁵ cells per well in 100 µl of culture medium followed by incubation for 4 h at 37 °C. Subsequently, cells were incubated with SYK inhibitors for 30 min followed by stimulation with Zymosan (final concentration of 50 µg ml⁻¹; Invivogen) and dispersed in culture medium for 24 h. Western blotting was performed using 15 μg of cell lysate and the following antibodies phospho-SYK-Tyr525/526 (1:1,000; Cell Signaling, catalog no. 2710), total SYK (1:1,000; Cell Signaling, catalog no. 2712) and b-actin (1:1,000; Proteintech, catalog no. 66009-1). For cytokine secretion quantification, cell culture medium was collected and concentrations of IL-10 in the supernatant were determined using mouse enzyme-linked immunosorbent assay kits (IL-10: Invitrogen) according to the manufacturer’s protocol. Experiments were performed in biological triplicates.

Cells were lysed using lysis buffer (50mM Tris HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 1mM EDTA) and denatured with Laemmli Sample Buffer (Biorad) at 95°C for 5 minutes. After SDS-PAGE, proteins were transferred to Immobilon-P Transfer Membrane (Millipore). After blocking with 5% BSA or 5% skim milk, blots were incubated overnight with following primary Abs: phospho-Smad1/5, phospho-Smad2, phospho-Smad3, phospho-Syk, total Syk, phospho-NF-κB p65, total NF-κB (all from Cell Signaling), or Actin (I-19) (SantaCruz). The blots were then incubated with HRP-Goat Anti-Rabbit IgG (H+L) (Zymed or Invitrogen), and bands were detected using ECL Select Western Blotting Detetion Reagent (GE Healthcare). Stripping was performed with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) or buffer prepared in-house (50 mM 2-ME, 2% SDS, 100 mM Tris-HCl).