Fig. 3. APP processing in CHO cell lines. (A) Endogenous full-length APP (upper panel) and APP-CTF (lower panel) were immunoblotted with APP C-terminal antibody (CT15) from the CHO cell line 13-5-1 deficient in LRP, the CHO cell line 14-2-1 expressing a mutant LRP defective in trafficking to the plasma membrane, and the control CHO K1 cell line. Note that cells lacking LRP (13-5-1) show a significant reduction in the level of APP-αCTF (bottom panel), while the cells expressing the LRP trafficking mutant (14-2-1) show higher endogenous APP-αCTF levels. (B) LRP is detected by immunoblotting with the polyclonal antibody 1704 raised against the C-terminus of LRP. As expected, no LRP can be seen in the CHO cell line 13-5-1 (middle lanes), while the amount of β-subunit in CHO 14-2-1 cells is significantly less than in control cells, with the ratio indicating lack of furin cleavage due to retention in the early secretory compartments. Uncleaved full-length LRP (α + β) can be visualized and the levels are comparable between CHO 14-2-1 and CHO K1 control cells. (C) Secretion of endogenous APPs into medium was increased in both CHO 13-5-1 and CHO 14-2-1 cells as compared with control CHO K1 cells.