Expression of transcobalamin/oleosin chimeric proteins in transfected N1E-115 cells.

A: Transgenic expression in N1E-115 cells at 48 h after transfection using lipofectamine. The cells were transfected with one of the following plasmids: pCMV-TCII-OLEO coding for transcobalamin-oleosin (lane 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (lane 2), pCMV-TCII coding for transcobalamin II (lane 3), pCMV-OLEO coding for oleosin (lane 4), pCDNA3 (lane 5), and pCMV-GFP-TCII-OLEO coding for GFP-TCII-OLEO (lane 6). The housekeeping gene was β-actin. The size in bp of the amplified products was 1347 for transcobalamin-oleosin (TCII-OLEO), 1240 for oleosin-transcobalamin (OLEO-TCII), 551 for transcobalamin II (TCII), 275 for oleosin (OLEO), and 349 for β-actin. B: Western blotting of homogenate of N1E-115 cells transfected with the various plasmids. From lane 1 to 6: homogenates from cells transfected with pCMV-TCII-OLEO, pCMV-OLEO-TCII, pCMV-TCII, pCMV-OLEO, empty plasmid, pCMV-GFP-TCII-OLEO, respectively. C: Vitamin B12 binding capacity in transfected cells. ⁵⁷Co-labeled Cobalamin (Cbl, ∼300 µCi per µg) was incorporated into culture medium (30,000 dpm/mL) for three days. The total amount of radioactivity taken by each cell lines was measured in pellets and supernatants. Mean and S.E.M. are indicated. D: Indirect immunofluorescence of TCII in N1E-115 cells transiently transfected with lipofectamine. The four constructs and the empty plasmid were tranfected in N1E-115 cells (1–5). The immunofluorescence was done with a goat polyclonal antibody to TCII and a donkey antigoat IgG fluorescein labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 10 µm. E: Confocal analysis showing co-localization of the protein GFP-TCII-OLEO with endoplasmic reticulum in transfected N1E-115 cells. The cells were transfected with the plasmid pCMV-GFP-TCII-OLEO coding for GFP-transcobalamin-oleosin (GFP-TCII-OLEO), using lipofectamine. Cell nuclei were counterstained with Hoechst 33258 (1, 5). Co-localization was evidenced with fluorescence from GFP (2, 6), immuno-fluorescence with a mouse monoclonal antibody to the human golgin-97 (3) or a rabbit polyclonal antibody to calreticulin (7) and merge fluorescence (4,8). The secondary antibodies include a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Calibration bars = 20 µm.