FIGURE 6.

Ifitm3 C termini are degraded in lysosomes. A, lysates from MEFs expressing an N- and C-terminally tagged Ifitm3 (myc-Ifitm3-C9) were examined by Western blot with staining for the indicated epitopes. Dotted lines indicate the sites of reassembly of the membrane after staining. B, Western blot for Myc-Ifitm3 and Myc-Ifitm3-LEDK and -REDI is shown at two exposures to highlight the double band pattern for Ifitm3-LEDK and REDI (upper) and confirm the lack of a second band for Ifitm3-KDEL (overexposed, lower). C, bafilomycin A1 treatment inhibits degradation of the C-terminal tag as assessed by Western blot. D, MEFs expressing Myc-Ifitm3-C9 were co-stained for Myc (green) and C9 (red) epitopes. The plasma membrane is yellow/orange, indicating co-localization of the two epitopes (full-length Myc-Ifitm3-C9). Green cytoplasmic puncta (arrow indicates an example) show the sites of removal of the C9 tag. The C9 tag remained intact within the enlarged vacuoles that accompany IFITM protein overexpression (arrowhead).