Figure 5

Mutations of RCC1 that remove the N-terminal tail, block N-terminal methylation or inhibit GEF activity do not prevent its interaction with apoRan. RCC1α wild-type (WT) and mutants (Δ27, K4Q and D182A) fused at the C-terminal with GFP-FLAG were expressed in U2OS cells (A) and immunoprecipitated with α-FLAG agarose beads (B). RCC1α-GFP-FLAG proteins were analysed by Western blotting using antibodies from Santa Cruz Biotechnology (sc-1162; does not recognize Δ27RCC1) (middle panel) and Transduction Laboratories (R35420) (bottom panel). The Transduction antibody detects both full-length RCC1α-GFP-FLAG and a lower band that is not detected by the Santa Cruz antibody. The latter therefore probably represents a truncated form lacking the N-terminal tail of RCC1α. A prominent non-specific band reacting with the Transduction antibody is indicated by *. Endogenous Ran present in the cell lysates (A) or co-immunoprecipitated with RCC1α-GFP-FLAG proteins (B) was detected by a specific antibody (top panel).