Biotinylated anti cd28 clone 37

Cells from spleen and LNs of CalDAG GEFI^–/–, CalDAG GEFI^+/– and CalDAG GEFI^+/+ mice were stained for dead cells using the LIVE/DEAD Fixable Dead Cell Stain (Invitrogen) and subsequently for surface markers CD4 and CD25. Next, cells were coated with 10 µg/ml biotinylated anti-CD3 (clone 145-2C11, BD Biosciences) and 5 µg/ml biotinylated anti-CD28 (clone 37.51, BD Biosciences) for 15 min on ice. After removal of excess antibody, cells were pre-warmed to 37 °C and stimulation was initiated by addition of 10 µg/ml streptavidin in cRPMI. After indicated time points, cells were fixed (BD Biosciences Phosflow Lyse/Fix) and permeabilized (BD Biosciences Phosflow Perm Buffer III) according to the manufacturer’s protocol. Cells were stained intracellularly for ERK1/2 pT202/pY204 or isotype control (both BD Biosciences Phosflow) and Foxp3 at 4 °C overnight.

Total CD4⁺ T cells were isolated from spleen and LNs of CalDAG GEFI^–/–, CalDAG GEFI^+/– and CalDAG GEFI^+/+ mice using mouse CD4 DirectBeads (L3T4) and the autoMACS separation system (Miltenyi Biotec). For stimulation, 1 × 10⁶ cells were resuspended in complete RPMI (cRPMI; 10% FCS, 25,000 Units penicillin/25 mg streptomycin, 1 mM sodium pyruvate, 25 mM HEPES, 50 µM β-mercaptoethanol) supplemented with either 20 ng/ml PMA and 1 µg/ml ionomycin (both from Sigma) or 35 µM sodium pervanadate (Sigma), and incubated for 5 min at 37 °C. For anti-CD3/CD28 stimulation, cells were coated with 20 µg/ml biotinylated anti-CD3 (clone 145-2C11, BD Biosciences) and 10 µg/ml biotinylated anti-CD28 (clone 37.51, BD Biosciences), and crosslinking was induced for 5 min by addition of 10 µg/ml streptavidin in cRPMI. As stimulation controls, cells were either left on ice or incubated at 37 °C for 5 min in cRPMI. Following stimulation, cells were washed once in ice-cold PBS, and cell pellets were further processed for lysis and Western Blotting.