Obesity-associated circulating factors promote greater Akt-mediated ERα phosphorylation and nongenomic ERα activity. Phosphorylation of ERα at two different sites (ser167 and ser118, the Akt and MAPK target sites, respectively) following a 15 minute or one hour exposure to 2% obese (Ob) or control (Con) patient sera was assessed in MCF-7 cells by western blot and standardized to tERα protein levels (A and B). The effect of tamoxifen (T) treatment on Akt and ERK1/2 activation in MCF-7 cells following a 15 minute exposure to 2% Ob or Con patient serum was also measured by western blot (C and D). Densitometry data from at least three independent experiments was compiled for each protein to calculate the average protein level, standard error of the mean and statistical significance, with one representative image for each protein shown. *, P < 0.05; **, P < 0.01 in comparison to Con, and different letters indicate significant differences (P < 0.05). ERα, estrogen receptor alpha.