Figure 6.

Rfp2 is a membrane-bound protein. (A) Schematic representation of selected RBCC proteins showing the varied C-terminal ends. (B) Top, U2OS cells were transfected with wild-type Rfp2 and with Rfp2-ΔTM, respectively. Forty-eight hours after transfection, cells were fixed in 4% formaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and stained with Rfp2 antibodies. Primary antibodies were visualized with FITC-conjugated anti-rabbit IgG, and F-actin fibers were visualized with Texas Red–conjugated phalloidin. DAPI was used to stain the nucleus. Bottom panel, HEK293 cell lysates transfected with Rfp2 and Rfp2-ΔTM were fractionated as described in Material and Methods, and fractions were analyzed by Western blotting for Rfp2 and a set of indicated marker proteins. Wild-type Rfp2 is localized to the membranous and nuclear fraction, whereas Rfp2-ΔTM is relocalized to the cytosol. Schematic figures represent the Rfp2 variants used in the transfections. (C) Subcellular fractionation of HEK293 followed by Western blotting for endogenous Rfp2 protein confirms the subcellular localization observed with ectopically expressed protein.