FIG. 6.

Properties of binding of rMYPE9110 and rN-MYPE9110^1-300 to mammalian cells. (A) Binding of full-length rMYPE9110 and rN-MYPE9110^1-300 determined by FACS analysis. Full-length rMYPE9110 and rN-MYPE9110^1-300 (both 250 nM) were incubated with 1 × 10⁶ HeLa cells for 30 min, followed by the addition of His antibody (1:500 dilution) and Alexa Fluor 488 (1:500 dilution). The representative histogram presents cell numbers plotted on the y axis and log fluorescence intensity plotted on the x axis. Cells incubated without protein were used as a control. (B) Comparative binding and internalization activities of full-length rMYPE9110 and rN-MYPE9110^1-300 determined by immunofluorescence. HeLa cells were incubated with no protein (control) (i) or intoxicated with either full-length rMYPE9110 (ii) or rN-MYPE9110^1-300 (iii) (250 nM) and analyzed by fluorescence microscopy. Cells were stained with DAPI (nucleus, blue), Alexa Fluor 633 (rMYPE9110, red), and Alexa Fluor 488 (phalloidin, green). All representative HeLa cell images are shown in the absence of ammonium chloride and at a ×400 magnification. (C) Localization of rMYPE9110 within intoxicated HeLa cells. Shown is a representative HeLa cell stained with DAPI (nucleus), Alexa Fluor 633 (rMYPE9110), and Alexa Fluor 488 (phalloidin). Digital orthogonal sections through z-series data sets (0.5 μm) confirmed that HeLa cell-associated rMYPE9110 is inside the cell. Images show HeLa cells intoxicated with rMYPE9110 and extended views of the horizontal (a) and vertical (b) z projections of HeLa cells. Arrows (white) indicate the localization of rMYPE9110 immunofluorescence in the horizontal and vertical z projections.