Figure 3 Confocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 μg/ml of human PD-L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes, fixed with 4% paraformaldehyde and stained with DAPI. Colocalization signal were examined with confocal microscope (scale bar = 2 µm)  Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001  Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours  Ability of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248 phosphorylated human PD-1 ITSM peptide was coated at 4 μg/ml in 96 plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours. Bound SHP2 were measured by monitoring the activity of horse-peroxidase conjugated on secondary antibody targeting SHP2 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001  Representative western blot showing the levels of total and phosphorylated ZAP70, PKCθ and PLCγ1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1μΜ of MB  Representative western blot showing the levels CD28 phosphorylation. CTL were stimulated with EG7 cells (parental), EG7-L1 and EG7-L1 in the presence 100 nM of MB for 2 hours. Protein immunoprecipitated (IP) with Anti-pY from the lysates of the indicated CTL-EG7 co-culture were subjected to SDS-PAGE separation and blotted with aCD28. β-actin served as loading control. EG7-L1: EG7 overexpressing PD-L1 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001  Phos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1μΜ MB. Cell lysate were then separated by SDS-PAGE containing 50 μM of Phos-tag and blotted with aCD2 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001