Protein analysis of CD28i in PBL-T cells and Jurkat T cells.

(A) SDS-PAGE analysis of HA-tagged CD28i protein in Jurkat T-cell transfectants. Stable transfectants of Jurkat T cells harboring CD28i-HA or N-linked glycosylation-deficient CD28i-HA were lysed in 1% Nonidet P40. Whole-cell lysates were fractionated by SDS-PAGE in reduced or nonreduced conditions and immunoblots were performed using anti-HA Ab. Arrows in lane 1 and lane 3 indicate CD28i-HA monomers. Arrows in lane 4 and lane 5 indicate N-linked glycosylation-deficient CD28i-HA monomers and CD28i-HA dimers, respectively. Lane 2 and lane 6 show samples of native Jurkat T cells. (B) The detection of CD28i protein in PBL-T cells and Jurkat T cells with CD28 C-terminal–specific Western blotting. The 1% Nonidet P40 cell lysates of PBL-T cells and Jurkat T cells were immunoprecipitated with a CD28 C-terminal–specific Ab (lanes 2 and 4) or the control Ab (lanes 1 and 3). Immunoprecipitates were fractionated by SDS-PAGE in the reduced condition and CD28 C-terminal–specific Western blotting was performed. Shown in lanes 5 and 6 are the data indicating the HA-tagged CD28i and HA-tagged CD28, which were visualized by HA-specific Western blotting. Lanes 1 and 2 are the data of human PBL-T cells, and lanes 3 and 4 are the data of Jurkat T cells. CD28 is indicated by an open arrow, and CD28i is indicated by a filled arrow in lanes 2 and 4. H indicates the position of the Ig heavy chain, and L indicates the position of the Ig light chain. The data represent 3 experiments with similar results.