Fig. 2.

Expression of transcobalamin-fused constructs in NIE115 cells. (A) The schematics of the plasmids TO, OT, and GTO are shown. (B) The incorporation of the three plasmids in NIE115 cells in corresponding stable cell line was evaluated by PCR technique (see Materials and Methods for the specific primers). (C) The relatively constant contents of the tyrosine hydroxylase in TO and OT cells indicate that these transfected cells remain as valid neuronal model (typical result). (D) Radiolabeled B12 (30,000 cpm at 300 μCi/μg) was added to ≈10⁶ cells and then incubated for 20 min at either 37 °C (intact cell) or 4 °C (lysed cell). Compared with OT, VR1, and WT, the expression of TO leads to an increased B12-binding capacity in either intact or lysed cells (P < 0.001). The VR1 expressing cells serve as a control to normalize the B12 binding to the TO, OT, and WT cells. (typical result out of five runs performed in three repeats. Mean and SE are indicated). (E) The confocal image of the NIE-115 cells transfected with GTO shows that the expressed fusion protein is targeted to reticulum (see supplemental figure for TO- and OT-tranfected cells). (F) Three days after initiating the differentiation of the NIE115 cells, more extensive neurite outgrowth was evident in TO cells. Here, cells were immuno-labeled with Adam17 antibody. The secondary antibody was coupled to Alexa Fluor 488 for visualization. P, proliferation; 3D, 3 days after differentiation; 5D, 5 days after differentiation; WT, wild-type NIE115 cells; BK, blank without DNA template; TC, transcobalamin.