Anti slp76py128

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.

The following antibodies were used for immunoblotting, cell-surface, and intracellular stains: The anti-LAT Y191 and anti-LAT from Millipore. The anti-LCK pY505, and anti-SLP-76 pY128, anti-IL-12Rβ1, and annexin V from BD Biosciences. The anti-PLC-γ1 pY783, anti-p38 pT180/Y182, anti-AKT pT308, anti-ZAP-70 pY319, anti-SRC pY416, anti-PLC-γ1, anti-LCK, anti-SLP-76, anti-AKT, anti-STAT4, anti-STAT4 pY693, anti-CD25, and anti-ERK 1/2 antibodies were purchased from Cell Signaling Technologies. The anti ERK1/2 pTpY185/187 and anti-AKT pS473 were from Invitrogen. The anti-GAPDH was from Meridian Life Science. The DyLight 800 and DyLight 680 labeled secondary antibodies were obtained from Thermo Scientific. The FITC anti-IFN-γ (4S.B3), Alexa Fluor 647 anti-IL-10 (JES3-9D7), Alexa Fluor 647 anti-IL-4 (8D4-8), Alexa Fluor 647 anti-TNF-α (Mab11), and APC anti-IL-13 (JES10-5A2) all from BioLegend. The anti-CD3 (OKT3), anti-CD4 (RPA-T4), FITC anti-CD28 (CD28.2), anti-CD-2 (RPA-2.10), anti-CD49d (9F10), anti-CD11a (HI111), FITC anti-CD278, anti-CD150, and DyLight 488 IgG (Ply4053) antibodies and propidium iodine were obtained from Biolegend. Anti-IL-12 Rβ2 was purchased from R&D Systems.