Figure 9

Secretion of Fam20a requires an intact signal sequence. A. Schematic representation of the putative signal sequence of Fam20a. The predicted cleavage site is indicated with a red arrowhead. The two amino acid substitutions introduced in the SSmut construct and the sequence remaining in the Δ23 mutant construct are shown. B. Immunoblot analysis of Fam20a and Fam20a(Δ23) protein levels in transfected COS-1 cells. The position of the glycosylated form of Fam20a (which is absent in Fam20a(Δ23) transfected cells is indicated with an arrowhead. C. Fluorescence images of COS-1 cells expressing either Fam20a-GFP or Fam20a(Δ23)-GFP. The wild type protein was observed within the cytoplasm, predominantly in a structure that is likely to be the ER. The mutant protein was primarily localized to the nucleus. D. Immunofluorescence images of Fam20a and Fam20a (SSmut) proteins as detected by antiserum directed against the C-terminal Myc epitope. The wild type protein was again detected in the ER and the mutant protein primarily in the nucleus. The cells have been counterstained with DAPI to delineate the nucleus.