Fig. 5. ERK1/2 is activated by LIF and protects against LIF/STAT3-induced apoptosis. (A) Western blot analysis of differentiated KIM-2 cells treated with 100 ng/ml LIF for the indicated times (30 minutes, 1 hour, 2 hours and 4 hours) in differentiation medium (DM). Immunoblotting was performed using specific antibodies against pSTAT3, total STAT3, pERK1/2 and total ERK1/2. The western blot is representative of three independent experiments. (B) Increased levels of activated ERK1/2 in mammary glands from involuting STAT3-deficient mice. Levels of phospho-ERK1/2 and total ERK1/2 in 2 day involuting glands from Stat3^-;/-; and Stat3^+/-; control mice was assessed by western blot analysis. Graphs indicate densitometry analysis for three independent Stat3^-;/-; mice, mean±s.e.m. (black bars) and two independent Stat3^+/-; mice (white bars). (C) Phase-contrast (left) and annexin V-FITC in situ fluorescence microscopy (right) of KIM-2 cells treated with 100 ng/ml LIF in the presence or absence of 10 µM U0126 for 24 hours (bottom row) and with U0126 alone (top row). MM, time-matched control in growth medium. The results are representative of three independent experiments.