Effect of epitope tag on hGnRH-RI signaling and spatial localization of FLAG-hGnRH-RI.

<p>(A) Inositol phosphate (IP) production in response to 100 nM Buserelin was assessed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011489#s4" target="_blank">Materials and Methods</a>”. Data represent three to six independent experiments performed in triplicate and normalized to FLAG-hGnRHRI ± S.E. (B, left) HTR-8/SVneo cells transfected with either FLAG-hGnRH-RI (a) subjected to indirect immunofluorescent staining using affinity purified rabbit anti-FLAG antibody followed by Alexa Fluor 568-conjugated anti-rabbit IgG (<i>red</i>) and counterstained with the nuclear dye, Hoechst (<i>blue</i>). Note the perinuclear localization of the FLAG-tagged hGnRH-RI (<i>a</i>, <i>arrow</i>); (B, right) HTR-8/SVneo cell transfected with GnRH-RI-GFP and counterstained with the nuclear dye, Hoechst (<i>blue</i>). Note the perinuclear localization of the GnRH-RI-GFP. (C) HTR-8/SVneo and HEK 293 cells transfected with FLAG-hGnRH-RI were subjected to indirect immunofluorescent staining using affinity purified rabbit anti-FLAG antibody followed by Alexa Fluor 568-conjugated anti-rabbit IgG (<i>red</i>) and counterstained with Hoechst (<i>blue</i>). Note the perinuclear localization of the FLAG-tagged hGnRH-RI seen in both cell lines (<i>arrows</i>). Bright field images are shown in the second column from the left. (D) Western blot analysis was performed on the lysates of nuclei isolated from HEK 293 cells overexpressing FLAG-GnRH-RI. The results reveal that the full length hGnRH-RI is expressed on the nuclei of HEK 293 cells. (E) Cells expressing FLAG-hGnRH-RI were subjected to indirect immunofluorescent staining for the receptor (red) as well as the nuclear membrane, endoplasmic reticulum and Golgi (<i>all shown in green</i>). Colocalization is seen as yellow staining. Column A: receptor alone; Column B: receptor + Hoescht; Column C: organelle marker (lamin A/C, calnexin or GM130) + Hoescht; Column D: receptor + organelle marker. Column letters and roman numerals used as a coordinate system. hGnRH-RI immunoreactivity co-localized with the nuclear marker, lamin A/C, as seen by the yellow staining (I-D, II-D, <i>arrows</i>) as well as with the endoplasmic reticulum marker, calnexin (III-D, IV-D). Less colocalization was seen between the receptor and the Golgi, as shown by less yellow staining (V-D, VI-D). Scale bar = 10 µm.