Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Diehn et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.092284399

Supporting Figure 9
Fig. 9.
Simultaneous engagement of CD28 enhances CD3-dependent dephosphorylation of NFATc1 and NFATc2. T cells were isolated and stimulated for 1 h with the indicated treatments, as described in Supporting Materials and Methods. Cells were lysed after the indicated times in RIPA (150 mM NaCl/20 mM Tris, pH 7.5/0.1% SDS/1% Triton X-100/0.5% sodium deoxycholate/1 mM EDTA) with protease and phosphatase inhibitors. Extract from 10⁶ cells was loaded per lane for SDS/PAGE on 7.5% gels. Westerns were probed with antibodies to NFATc1 (monoclonal 7A6, Crabtree lab) or NFATc2 (polyclonal, S. Stewart, Stanford University), and the phosphorylation state of the bands is as indicated. Dephosphorylated forms of NFATc1 migrate as "smears" between and below the three phosphorylated forms.