Fig. 2.

The ICOS transmembrane domain mediates calcium costimulation and is required for p85 recruitment. a Surface ICOS staining of Jurkat cells stably expressing ICOS, ICOS-CD44TM, or ICOS-NT molecules. b Calcium mobilization in Jurkat cells expressing ICOS molecules of the indicated types after stimulation with biotinylated anti-CD3 (0.2 μg/ml) and anti-ICOS (2 μg/ml) antibodies coligated by streptavidin. The data represent the results of three independent experiments. c Immunoblotting of phospho-ZAP70 and phospho-PLCγ in Jurkat cells expressing the indicated ICOS molecules 30 s after stimulation with anti-CD3 (0.2 μg/ml) and anti-ICOS (2 μg/ml) antibodies coligated by streptavidin. The data represent the results of three independent experiments. d Input (3% total lysates) or anti-ICOS immunoprecipitates of the indicated Jurkat cells before or 1 minute after ICOS crosslinking stimulation were probed by Western blotting for p85 and ICOS. e Immunoblotting of the indicated molecules in ICOS or ICOS-CD44TM Jurkat cells following anti-ICOS crosslinking stimulation for the indicated periods of time. Left, representative pAkt patterns; right, normalized pAkt levels of three independent experiments