Fig. 5.

Sec49K binds to the cell surface phosphatase CD45. (A) Jurkat and its CD45⁻ derivative J-AS-1, as well as two J-AS-1 transfectants expressing the CD45R0 and CD45RABC isoform, respectively, were incubated with medium (Upper) or sec49K (Lower). After washing, cells were stained for CD45 using mAb HI30 (black histograms) followed by Alexa Fluor 488-labeled goat anti-mouse IgG (Invitrogen). In parallel, cell-bound sec49K was detected by mAb 4D1, followed by Alexa Fluor 488-labeled goat anti-rat IgG (gray histograms). The background staining obtained with the secondary reagents only is shown in white. The results represent one of three similar experiments. (B) 293T cells were transiently transfected with expression constructs for CD45RO, CD45RABC, or control Ad2 E3 DNA as indicated on the right. At 40 h later, cells were incubated with either BSA or sec49K, after which CD45 expression and sec49K binding were monitored using single staining (α49K; 4D1) or double staining with Tricolor-labeled anti-CD45 mAb HI30 (α49K+CD45) and mAb 4D1. The data represent one of three similar experiments. (C) Ad19a E3/49K coimmunoprecipitates CD45 expressed in Jurkat cells. Jurkat cell lysates (J) were incubated with cell lysates of 293 cells (293) and 293 cells stably expressing E3/49K (K35; 0.5 mg each). Immunoprecipitations (IP) were performed using 1 μg of mAb MEM28 recognizing CD45 (CD45) or 4 μg mAb 4D.1 recognizing E3/49K (49K). The precipitated proteins were evaluated by Western blot analysis (WB) with the same antibodies. The data are from one of two representative experiments.