Journal: Journal of cell science

Article Title: Improperly folded green fluorescent protein is secreted via a non-classical pathway.

doi: 10.1242/jcs.00047

Figure Lengend Snippet: Fig. 1. GFP is secreted via a non-classical pathway in CHO cells. (A) Untransfected CHO cells (Ctrl) and cells transiently transfected with either GFP (GFP) or preprolactin-myc (PPL) were labeled with [35S]-methionine and chased for 6 hours. The cell lysates (C) and media (M) were subjected to immunoprecipitation with either antibodies against GFP (lanes 1-8) or myc (lanes 9-12), and the washed immunoprecipitates were displayed by SDS-PAGE and fluorography. Some cells (+BFA) were treated with 1 µg/ml brefeldin A for 1 hour prior to labeling (lanes 3- 4, 7-8 and 11-12). ‘% Sec.’ denotes the percentage of secretion of GFP into the media as determined by a phosphorimager. (B) CHO cells transfected with plasmids encoding either mouse dihydrofolate reductase (mDHFR) or Schistosoma japonicum glutathione S-transferase (GST) both tagged with the FLAG epitope. The cells were labeled with [35S]-methionine and chased in serum-free medium. The cell lysate (C) and media (M) were immunoprecipitated with anti-FLAG antibodies and processed as above. (C) Plasmids coding for mDHFR and GFP were used to co-transfect CHO cells. The cells were labeled, chased and processed as described in B.

Article Snippet: Polyclonal antibodies against the FLAG epitope (DYKDDDDK) were purchased from ProSci.

Techniques: Transfection, Labeling, Immunoprecipitation, SDS Page, FLAG-tag