FIGURE 4.

Expression of CLEC2D protein isoforms in transfectants. 293T cells were transfected with CLEC2D isoform 1 (LLT1) alone or together with CLEC2D isoforms 2 and 4 or with CLEC2A. A, LLT1 alone was detected by Western blot analysis of whole cell lysates using 2F1 mAb under reducing conditions. β-Actin was used as internal control. B, CLEC2D isoforms 2 and 4 were detected on whole cell lysates using anti-Myc 9E10 mAb. A nonspecific band was detected in all samples at ∼38 kDa. C, supernatants of transfected COS cells were analyzed for the presence of soluble LLT1 by slot-blot using anti-Myc 9E10 mAb. D and E, cell surface expression of CLEC2D protein isoforms was analyzed by flow cytometry on 293T cells (D) or 293T-LLT1 cells (E) transfected with CLEC2D isoforms or CLEC2A. LLT1 expression was monitored using anti-LLT1 4F68 mAb and the other CLEC2D isoforms and CLEC2A using anti-Myc 9E10 mAb. F, CLEC2D protein isoform expression on whole cell lysates following Endo H or PNGase F treatment. G, LLT1 expression after immunoprecipitation of CLEC2D isoforms 2 and 4 with anti-Myc, revealed using 2F1 mAb. The white arrow indicates monomers of LLT1 under reducing conditions, and asterisks indicate the heavy and light chains of the anti-Myc mAb used to immunoprecipitate. The results are representative of two to four independent experiments.