Figure 3

A, Summarized data indicating effects of CPE on DAT membrane expression in cotransfected HEK-293 cells. Columns show the means ± SEM of the ratios of colorimetric readings under coexpression of DAT and pcDNA3 conditions versus coexpression of DAT and CPE. **P < 0.01, n = 3 (Student's t test). B, Summarized data indicating effects of CPE on DAT total expression including the extracellular and intracellular ones in cotransfected HEK-293 cells. Columns show the means ± SEM of the ratios of colorimetric readings under coexpression of DAT and pcDNA3 conditions versus coexpression of DAT and CPE. **P < 0.01, n = 3 (Student's t test). C, CPE colocalizes with DAT in HEK-293 cells. The distribution pattern of DAT and CPE when expressed individually or coexpressed in HEK-293 cells are shown. Immunostaining was performed using the rabbit anti-DAT or the mouse anti-CPE antibodies and secondary antibodies:CY3 anti-mouse (red) and FITC-conjugated anti-rabbit antibodies (green). Yellow staining on the merged panels depicted colocalization. No staining was observed when primary antibodies were eliminated. Data are representative of 4–6 independent experiments, with a total cell sampling of more than 1000. D, Measurement of DAT protein metabolism by labeling with radioactive amino acids. HEK-293 cells, into which the indicated genes had been transfected, acutely incorporated 0.2 mCi/ml promix [³⁵S]-methionine/cysteine for 4 h and were grown in the complete medium for further 6, 12 and 24 h. DAT protein was immunoprecipitated from the total protein by an anti-DAT polyclonal antibody. The antigen-antibody complex was further analyzed on polyacrylamide gel and autoradiography was performed. The results were repeated 3 times.