Figure 2

Phospho-epitope profiling of signaling cascades upon LFA-1 signaling. (A) Graphical representation of screening strategy. (B) 250,000 naïve CD4+ T cells were crosslinked with CD3 (mAb UCHT1), CD28 (clone 28.2) or ICAM ligand on U-bottom plates for 5, 10, 15, 30 minutes. Adherent cells were fixed, permeabilized, and stained for phospho-epitopes. Data values are relative to non-adherent control cells and were computed using Spotfire software. Data is displayed as a scattered plot profile as a function of the concentration of stimulation agent. Data is averaged from triplicate measures from four individual donors. (C) Phospho-proteomic analysis of p38 and zap70 after 5 minutes stimulation at 1000 ng ligand. Data was computed as the ratio of stimulated to unstimulated cells. Error bars are computed from triplicates. (D) Ratiometric measure of intracellular phosphorylated-p44/42 and pan-p44/42 as a function of CD3/CD28, ICAM-1/CD3/CD28, ICAM-2/CD3/CD28, or ICAM-3/CD3/CD28 stimulation (1000 ng) after 15 minutes. Cells were stimulated in 96-well coated plates and then stained with CD4-PercpCy5.5, phospho-p44/42-PE, and pan-p44/42-AX680. Quantitation of the ratio of phospho-p44/42 to pan-p44/42 was computed in Flowjo. The data is representative from triplicate experiments. The experiment was performed using cells from two donors.