p85β overexpression enhances CBL-b and TCR down-regulation. (A) TCR down-modulation was measured by flow cytometry in purified peripheral CD8⁺ T cells from p85β^−/− and p85β^+/− F5TCRTg mice stimulated in vitro with CD3 or CD3+CD28 for the times indicated. TCR down-regulation was estimated as the reduction in the mean fluorescence index of TCRβ expression on stimulated CD8⁺ versus nonstimulated CD8⁺ T cells (mean ± SD, n = 3). (B) Cells were activated as in panel A, and Nedd4 levels were examined in WB. The graph quantitates the mean plus or minus SD (n = 3) of Nedd4 signal relative to maximal levels (100%). (C,D) Jurkat T cells were transiently transfected with empty vector or rHAp85α or β. Cells were activated as in panel A using CD3+CD28 36 hours after transfection and then collected. We analyzed CBL-b and c-CBL levels in cell extracts by WB (C) or TCR down-modulation as in panel A (D); MFI of TCRβ expression on stimulated CD4⁺ versus gated nonstimulated CD4⁺ T cells (mean ± SD, n = 4; D). Graphs (C) show CBL signal intensity in AUs (mean ± SD, n = 3). (E) Purified peripheral CD8⁺ T cells from p85β^−/− and p85β^+/− F5TCRTg mice were stimulated in vitro with peptide-pulsed APCs. Cell extracts were examined in WB (indicated). The graphs represent the mean plus or minus SD (n = 3) of the signal for each of the proteins in 3 different assays, compared with the maximal signal (100%) in WT cells at 15 minutes (pPKC), 30 minutes (pPKB), or 1 hour (p-p38, p-p44/42) and normalized in comparison with loading controls (n = 3). P values are shown for the time points of maximal signal in WT cells (*P < .05).