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. 2015 Dec 7;291(5):2246–2259. doi: 10.1074/jbc.M115.686964

FIGURE 2.

FIGURE 2.

Quantifying GnRHR-mediated ERK and NFAT signaling in HeLa and LβT2 cells. Panel A, HeLa cells (open bars) were transduced with Ad mGnRHR and Ad Egr1 zsGREEN and treated with 0 or 10−12–10−6 m GnRH before staining, imaging, and calculation of I(ppERK;GnRH) from 5-min data and I(Egr1;GnRH) from 360-min data as described under Fig. 1. Alternatively, they were treated with Ad m-GnRHR and Ad NFAT1c-EFP and Ad NFAT-RE asRED and stimulated with 0 or 10−12–10−6 m GnRH before staining and imaging. Whole cells asRED and the nuclear fraction of NFAT1c-EFP (NFAT-NF) were calculated for each individual cell, and these values were used to calculate I(NFAT-NF;GnRH) and I(NFAT-RE;GnRH) using 20- and 360-min data, respectively. Parallel experiments were performed with LβT2 cells (filled bars) with identical procedures except that the LβT2 cells express endogenous GnRHR and, therefore, did not receive Ad-mGnRHR. The I(response;GnRH) values shown are the means (±S.E., n = 3). Panel B, HeLa cells transduced with Ad-mGnRHR were stimulated with 0 or 10−12–10−6 m GnRH and processed as described above except that Ad m-GnRHR titer was varied (0, 0.325, 0.625, 1.25, 2.5, 5, and 10 pfu/nl). The I(ppERK;GnRH) values shown are the means (±S.E., n = 3) and are plotted against GnRHR number estimated as described under “Experimental Procedures.”