RHAMM is not a cell surface but an intracellular cytoskeletal protein in mouse embryonic stem (ES) cells.

<p>(A) Confirmation of the gene trap insertion and expression levels of RHAMM and Nudcd2 in parental (E14TG2; E14) and RHAMM^+/- (BB0166; BB) mouse ES cells were measured by RT-PCR. GAPDH served as an internal control. Gene expression of RHAMM was reduced in RHAMM^+/- cells whereas <i>Nudcd2</i> was unaffected by the gene trap insertion. (B) Western blot analysis revealed a marked reduction of RHAMM protein in RHAMM^+/- mouse ES cells (BB0166). Similar reduction of RHAMM was observed in an alternate RHAMM^+/- mouse ES cell-line, XP0038. Actin served as a loading control. (C) Fluorescence activated cell sorting (FACS) of non-permeabilized (left panel) mouse ES cells failed to detect extracellular RHAMM using a C-terminal directed antibody. Similar results were obtained with an N-terminal directed antibody (not shown). Relative to negative control secondary antibody only, both cell-lines were strongly positive for SSEA-1. Alcohol permeabilized mouse ES cells, however, were strongly positive for both RHAMM and the intracellular positive control protein TPX2 (right panel). (D) Immunofluorescence detection of RHAMM in RHAMM^+/+ mouse ES cells co-localized with microtubules (acetylated tubulin; ac-tubulin) and cell-cell junctions (ZO-1). Arrows indicate co-localization with mitotic spindles, while double arrows indicate co-localization with mitotic centrosomes. Scale bar: 20 µm. (E) RHAMM^+/+ mouse ES cells grown without feeders were fixed in 4% paraformaldehyde and stained with antibodies to either RHAMM or beta-tubulin. The arrows point out mitotic spindles that stain positive despite the lack of detergent in the fixation procedure. Addition of detergent (0.1% Triton X-100) to the methodology results in significantly better penetration of the antibodies and amplification of the detected signal. Scale bar: 10 µm.