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. Author manuscript; available in PMC: 2011 Sep 27.
Published in final edited form as: Cancer Res. 2008 Dec 1;68(23):9712–9722. doi: 10.1158/0008-5472.CAN-08-1952

Figure 3.

Figure 3

Combined treatment with leptin and IGF-I leads to a synergistic increase in phosphorylation of both Akt and ERK in breast cancer cells. MDA-MB-468, MDA-MB-231, and MCF-7 cells were serum starved for 24 h, followed by treatment with 100 ng/mL leptin, 100 ng/mL IGF-I alone, and the combination for 30 min. Total protein was isolated from MDA-MB-468, MDA-MB-231, and MCF-7 cells and equal amounts of proteins were subjected to immunoblot analysis with specific antibodies against phospho-Akt (p-Akt) and Akt (A) or phospho-ERK (p-ERK) and ERK (B). Increased phosphorylation of Akt was observed in MDA-MB-468, MDA-MB-231, and MCF-7 cells treated with leptin or IGF-I, whereas combined treatment induced further increase in Akt phosphorylation. Leptin and IGF-I treatment increased phosphorylation of ERK, whereas combined treatment induced a synergistic increase in ERK phosphorylation. The membranes were reblotted with ERK, Akt, and anti-actin antibodies as control. Representative blots of multiple independent experiments. The representative histogram is the densitometric analysis of the Western blot signals showing fold increase in levels of phospho-Akt and phospho-ERK with respect to total Akt and ERK. #, P < 0.01, compared with untreated controls; *, P < 0.05, compared with untreated controls; **, P < 0.01, compared with leptin-treated cells.