Breast cancer cell Akt, ERK1/2, and IGF-1R activation are enhanced by obesity-associated circulating factors. The effects of obese (Ob) and control (Con) patient sera on Akt, ERK1/2 and IGF-1R activation were assessed by western blotting. MCF-7 (A and B) and T47D (C and D) cells were exposed to 2% Ob or Con patient sera for 15 minutes or one hour; pAkt (ser473) and pERK1/2 protein levels were then measured and standardized to tAkt and tERK1/2 protein levels, respectively. (E and F) pIGF-1R (tyr1135/1136) protein levels in MCF-7 cells were measured and standardized to tIGF-1R following a 15 minute incubation in 2% Ob or Con patient sera. Densitometry data from at least three independent experiments were compiled for each protein and cell line 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. IGF-1R, insulin-like growth factor 1 receptor.