FIG. 1.

Purification of K1-GST protein and antibody production. (A) Purification of K1-GST protein. Supernatants from 293T cells transfected with expression vector pDEF3-K1-GST-AU1 were harvested and run over a glutathione Sepharose resin. Purified proteins were stained with Coomassie blue (top) or subjected to immunoblotting (IB) with an anti-GST antibody (αGST) (bottom). The first lane shows molecular weight (MW) standards, and lanes 2 to 11 contain the K1 proteins from 10 different purifications. (B) Flow cytometry analysis. Surface expression of K1 on BJAB cells was assessed by staining with preimmune mouse serum (dotted line) or anti-K1 antibodies, followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody. The level of antibody reactivity (high, medium, or low) is indicated. (C) Immunoblot assays. Whole-cell lysates were used for immunoblotting with anti-K1 antibody 2H5. Lanes: 1, 293T cells transfected with pDEF3-GST; 2, 293T cells transfected with pDEF3-K1-GST; 3, 293T cells; 4, Jurkat-T cells; 5, BJAB cells; 6, BJAB-Flag-K1 cells; and 7, BJAB-K1-His cells. The arrows indicate K1 and K1-GST proteins. (D) Immunofluorescence test. BJAB cells expressing the Flag-tagged K1 were fixed, permeabilized, and reacted with anti-Flag or anti-K1 antibodies (3A4, 3C12, 2H5, and 3A3), followed by Alexa 486-conjugated goat anti-mouse IgG. Cells were additionally stained with To-Pro3 solution (blue) for 1 min to show the nucleus. Immunofluorescence was examined with a Leica confocal immunofluorescence microscope, and a single representative optical section is presented.