Figure 5

Receptor activator of NF-kB (RANK)-c reduces the levels of wild type (wt) RANK on the cell surface. A. 293T cells transiently transfected with wt HA-RANK in combination with green fluorescent protein (GFP)-RANK-c. Cells were fixed and stained with the use of triton 0.01% for permeabilization of the cell membrane. Representative recording of a merged field (upper panel) showing the membrane localization of a single-positive human influenza hemagglutin epitope (HA)-RANK (red) cell and the cytoplasmic retention of HA-RANK when co-expressed with GFP-RANK-c. In the lower panel the split field of the same field in more detail. White boxes indicate the area of focus. The arrowheads depict the clear membrane localization of wt RANK when expressed alone in a cell. B. Four representative fields of unpermeabilized (non triton) cells fixed and stained as above. In the capture only merged fields are depicted. Split channels can be found in Additional file 6. HA molecules were visualized with the use of anti-HA from Santa Cruz (sc-57592). C. 293T cells were transfected with HA-wt RANK alone or in combinations with RANK-c and analysed by flow cytometry. Isotype control from mock-transfected cells (grey-filled) wt RANK is depicted by the red line and co-transfections of wt RANK and increasing amounts of RANK-c (0.25 and 0.5μg) are depicted by blue and yellow lines, respectively. RNA was extracted from transfected cells after flow cytometry and mRNA expression levels of the indicated isoforms were quantified by PCR (right panel).