Fam20a is a secreted protein. COS-1 cells were transfected with either an empty expression vector (-) or one encoding mouse Fam20a with a C-terminal myc epitope tag and proteins were isolated from either the medium (panel A) or the cells (panel B). The proteins were analyzed by immunoblotting using a Myc tag-specific antiserum. Samples in lanes 2 and 4 of each blot were pre-treated with protein N-glycosidase prior to analysis to remove glycosyl groups. A recombinant form of Fam20a synthesized in rabbit reticulocyte lysates (TnT) was included on each gel as a size marker. The position of glycosylated and deglycosylated Fam20a is indicated using arrowheads and cross reacting material detected in the medium is indicated using asterisks. The location of molecular size markers is shown on the left of each gel. C. Protein samples from the medium of transfected cells that were untreated or treated with Brefeldin A were analyzed by immunoblotting using the Myc tag-specific antiserum. As Brefeldin A was resuspended in DMSO, the untreated cells were exposed to DMSO alone as a vehicle control. The amount of Fam20a detected in the medium of Brefeldin A treated cells was consistently lower than that observed in untreated cells (indicated using an arrowhead).
Nalbant et al. BMC Genomics 2005 6:11 doi:10.1186/1471-2164-6-11