FIG. 11.

Nuclear translocation of the IFNaR2 ICD. (A) Nuclear translocation detected by indirect immunofluorescence. U5A cells expressing a reverse-tet transcriptional activator and a tetO-regulated IdelE-HA construct were plated on glass coverslips, and either induced (+DOX) (50 ng/mL) for 24 h or left uninduced (−DOX), lysed in situ (see Materials and Methods), fixed, and incubated with anti-HA antibody followed by an Alexifluor-594-labeled secondary antibody. Nuclear counter-staining was performed with Hoechst 33258. Representative cells visualized under a fluorescent microscope are shown. These images are representative of 3 replicate experiments. (B) Nuclear translocation detected by immunoblotting. Plasmids encoding constitutively expressed full-length IFNaR2-HA (FL) or IdelE-HA were transiently transfected into HEK293T cells, harvested 24 h later, swollen in hypotonic buffer, lysed in 0.3% NP40, and centrifuged. The supernatant was used for non-nuclear fractions and the pelleted nuclei were extracted with high salt to obtain nuclear proteins. The protein fractions were immunoblotted with anti-HA. Each lane contains lysate from the same number of cells. These immunoblots are representative of 2 replicates. Non-nuc, non-nuclear.