Slp 76

Concanavalin A, actinomycin D and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) were obtained from Sigma-Aldrich; mouse or human TNF-α was from PeproTech; α-galactosyl ceramide (α-GalCer; KRN7000) was from Enzo Life Science; R788 was from Cayman Chemical.
Antibodies to IκBα, p-ERK, p-JNK, p-p38 (T180Y182), p-MKK3/6, p-MKK4, SYK, PLCγ1, Vav-1, SLP-76, PKCθ, cleaved caspase 3, caspase 9, and cleaved caspase 9 were purchased from Cell Signaling Technology; anti-p-p38 (Y323) was from ThermoFisher Scientific; anti-GAPDH Ab was from Chemicon; Abs to mouse CD3, CD28, and Zap70 were from Biolegend; anti-CD3-FITC, anti-CD4-PE, anti-NK 1.1-PE, and anti-CD69-PerCP Abs, and Annexin V-FITC apoptosis detection kit were from eBioscience; anti-Gadd45α Ab was from Santa Cruz Biotechnology.

Cells were harvested by centrifugation at 120 g for 5 minutes in a microfuge. The pellets were washed with PBS and lysed with mammalian protein extraction reagent (Pierce) with protease and phosphatase inhibitors (Sigma). The lysates were frozen at −80°C overnight and defrosted on ice, then cleared by centrifugation at 16 060 g for 10 minutes at 4°C. The supernatants were collected, and protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of total protein lysates (30 μg) were resolved by SDS‐PAGE, electrotransferred onto PVDF membranes, probed with antibodies, and detected with ECL reagent (GE Healthcare). The antibodies used for western blots are as follows: IRF‐1 was from BD Biosciences (San Jose, CA); GAPDH, c‐Raf, Lyn, Fgr, p47phox, Slp‐76, Vav1, PU.1, horseradish peroxidase anti‐mouse, and anti‐rabbit antibodies were from Cell Signaling (Danvers, MA); and c‐Cbl was from Santa Cruz Biotechnology (Santa Cruz, CA). The relative intensity of specific band was calculated against GAPDH using ImageJ software.