Relevance of GnRHR expression level for TEY phosphorylation unattributable nuclear localization of ERK2-GFP.

<p>For panels A and B, HeLa cells transfected with ERK siRNAs were transduced with Ad ERK2-GFP (WT) and with Ad mGnRHR at varied titre (0, 0.03, 0.06, 0.125, 0.25 or 0.5 pfu/nl) in order to vary GnRHR number. They were then stimulated for the indicated period before being fixed and stained for ppERK2-GFP and DAPI. Imaging and analysis of single cells was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040077#s2" target="_blank">Methods</a>. The plots show cell population averaged ppERK2-GFP levels for cells stimulated with 100 nM GnRH plotted against time (A), or for cells stimulated 15 min with 0 (Ctrl.) or 100 nM GnRH, plotted against Ad mGnRHR titre (B). These data are from a single experiment with duplicate wells, and are representative of those from 2 similar experiments. Panel C show ERK2-GFP N:C ratios calculated for data from the same experiment, binned according to ppERK2-GFP levels (80 AFU per bin, accepting a minimum of 50 cells per bin in each experiment). Data are shown for cells transduced with 0.25 pfu/nl Ad mGnRHR and stimulated for 0 (Ctrl.), 15 or 30 min with 100 nM GnRH. Panel D shows ppERK2-GFP values (AFU) and ERK2-GFP N:C ratios calculated for cells within a single ppERK2-GFP bin (120–160 AFU), plotted against Ad mGnRHR titre. ANOVAs of the ppERK2-GFP data in panels B and C both revealed Ad mGnRHR titre as a significant variable with significant elevation (P<0.01 compared to control without Ad mGnRHR) at all titres.