Characterization of PC12 cells stably expressing HA-ApoER2. (A) Schematic representation of the human HA-ApoER2 receptor transfected into PC12 cells. (B) Western blot showing the expression of HA-ApoER2 in stably transfected PC12 cells but not in wild type cells. ApoER2 was detected using an antibody that recognizes a region near the C-terminus of ApoER2. Both the mature (glycosylated) and immature forms (~170 and 130 kDa) of the receptor were detected. There are also recognized fragments close to 26–34 kDa and 17 kDa (corresponding to the receptor C-terminal fragment, CTF). α-tubulin is shown as a loading control. (C) Immunofluorescence of PC12 cells transfected with HA-ApoER2 (red) under basal conditions and after 72 h of NGF treatment (100 ng/mL) to induce differentiation. The cells expressed the receptor in different regions, including the plasma membrane and growth cones. Nuclear staining is shown in blue. Scale bar: 20 μm. (D) Western blots of cell lysates from PC12 cells stably expressing ApoER2. AKT phosphorylation was still detected after 2 h of incubation with NGF (100 ng/mL).