(A) EMSA analysis of nuclear proteins bound to the M67 hSIE DNA probe after MHC-I or TCR/CD3 ligation. Cells (10⁷) were preincubated with PBS (lane 1) or saturating amounts of anti-β₂M Ab (lanes 3 through 5) before exposure to avidin for the indicated time (lanes 1 and 3 through 5) or exposed to anti-TCR/CD3 Ab for 30 minutes (lane 2). Nuclear extracts were subjected to EMSA analysis as described in the Materials and Methods. The shifted DNA was blocked with 50-fold excess nonlabeled DNA (lane 5). (B) The EMSA analysis was performed as described under (A), but the nuclear extract was preincubated for 15 minutes with antibodies before the addition of the labeled M67 probe. Experiments shown in lanes 1 and 2 were preincubated with anti–Stat-3 antibody and those shown in lanes 3 and 4 with isotype control antibody (anti–ISGF-3γ). (C) Precipitation of Stat-3 from the nuclear extract. EMSA nuclear extract was made as described under (A) and precipitated with a biotinylated M67 probe and avidin-coupled agarose as described in the Materials and Methods. Cells (10⁷) were preincubated with PBS (lane 1) or saturating amounts of anti-β₂M Ab (lanes 2 through 4) before exposure to avidin for the indicated time (lanes 1 through 4). The precipitate were immunoblotted with anti–Stat-3 Ab.