Fig. 6
p150 is released from the plasma membrane by ectodomain shedding. a Two DPF constructs were engineered. One has an N-terminal FLAG (see Fig. 4a) and the other a C-terminal FLAG. b Cells expressing N-FLAG and C-FLAG were shaken in DB for 18 h and media and membrane fractions prepared and immunoblotted to α-FLAG. The most abundant N- and C-terminal tags are localized to separate sized fragments, suggesting processed cleavage for ectodomain shedding. A full-length DPF form is in the membrane as p160; it is processed to release p150 and membrane-anchored p10 (see Fig. 6a). c A C-terminal GFP DPF protein expression construct was also engineered. d Media and membrane fractions from WT cells and WT cells expressing C-GFP (C-GFPOE). Media proteins were immunoblotted to α-DPF (see Fig. 4a,c and Additional file 3: Figure S3C), and membrane proteins were immunoblotted to α-GFP. α-DPF detects secreted p150 and α-GFP detects membrane-anchored p10 fused to GFP. e Fluorescence localization of GFP in C-GFP expressing cells. Strong GFP fluorescence, as a read-out of the DPF TM domain, is seen at the cell periphery