Figure 5.

NFX1-91 is ubiquitinated by E6-independent and E6-dependent means. (A) Western blot. 293Ts were transiently transfected with Flag-tagged forms of NFX1. Cells were treated with MG-132 prior to lysis. Proteins were detected with rabbit anti-Flag antibody. (B) The lysates shown in A were immunoprecipitated with rabbit IgG (rIgG) or a rabbit anti-Flag antibody (α-Flag). Western blots with a mouse anti-ubiquitin antibody indicate that NFX1-91 was highly ubiquitinated, whereas NFX1-123 was not. (C) Endogenous NFX1 isoforms were immunoprecipitated from HFK/LXSN and HFK/E6 cells with antibodies specific for each isoform or with preimmune serum. Anti-NFX1 detects both NFX1 isoforms and anti-ubiquitin detects ubiquitinated proteins. Nucleolin is a loading control for the input lysates.