Identification and Characterization of a Novel Nontranslated Sequence Variant of the Human Intestinal Di-/Tripeptide Transporter, hPEPT1
Figure 3
Western blot and immunolocalization analysis of hPEPT1-RFI, hPEPT1, or mock transfected HEK 293 cells. (a) The anti-HIS antibody did not detect any difference between cell lysates from hPEPT1-RFI myc/HIS expressing and mock cells (10 and 11 μg) (lane 2 and 3). The positive control Positope protein, containing the HIS epitope, was detected (53 kDa) with the anti-HIS antibody (lane 1). M denotes the molecular weight marker. (b) Western blot on cell lysates from hPEPT1 expressing HEK 293 cells. The anti-hPEPT1 antibody detected the hPEPT1 protein (80 kDa) (lane 2 and 3) (3 and 10 μg), but as expected not in the mock transfected cells (lane 1) (10 μg). Cells transfections were performed in parallel for the hPEPT1-RFI, hPEPT1, and mock-containing vectors. (c) Immunostaining of cells transfected with the myc/HIS tagged hPEPT1-RFI, or mock plasmid (d) using the monoclonal antibody anti-myc-FITC antibody (green). Cells were counterstained with propidium iodide to visualize cell nuclei (red). (e) Immunostaining of cells expressing hPEPT1 with an anti-hPEPT1 antibody followed by a secondary alexa 488-conjugated antibody. Immunostaining was completed in two independent cell passages. The image is representative of three individual preparations. Immunostaining against the HIS epitope confirmed these findings in two independent cell passages.
