Figure 2

CHO-K1 cells do not express NPFF receptors. The presence or absence of NPFF receptors was analyzed with immunocytochemistry, RT-PCR and radioligand binding assay. A. CHO-K1 cells expressing hNPFF2R showed intense fluorescence when immunolabeled with an anti NPFF2R antibody detecting the C-terminus of the receptor. B. In the same conditions, the C-terminally oriented antibody did not label nontransfected CHO-K1 cells. C. The NPFF2R expression was also studied with an anti-NPFF2R antibody that binds to the N-terminus of the receptor. Again, in CHO-K1 cells expressing hNPFF2R immunoreactivity for the receptor was found. D. The N-terminally oriented NPFF2R antibody did not detect NPFF2R immunoreactivity in the nontransfected CHO-K1 cells. All the images are 0.5 μm confocal sections at the mid-nuclear level. Representative images are shown. Scale bar 20 μm. RT-PCR analysis gave further support for the absence of the NPFF receptors in CHO-K1 cells. E. cDNA from CHO-K1 cells were analyzed with hNPFF1 receptor specific primers and no PCR-product was obtained; human hypothalamus was used as a positive control (expected size of the PCR-product ~350 bp). F. The RT-PCR analysis of CHO-K1 cDNA did not give any PCR-product with hNPFF2R specific primers either; human placenta was used as a positive control (expected size of the PCR-product ~450 bp). G. CHO-K1 cells did not bind the NPFF receptor-specific radioligand at the concentration range where the positive control cell line expressing hNPFF2R showed saturable binding. The solid squares represent the total binding, the solid circles the nonspecific binding and the solid triangles the specific binding to the CHOK1 cell membranes. H. The cell membranes from CHO/hNPFF2R cells were used as a positive control in the experiment. The open squares represent the total binding, the open circles the nonspecific binding and the open triangles the specific binding to the cell membranes.