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The reactive bands were visualized with ECL plus reagents as previously described.

The protein bands were visualized by enhanced chemiluminescence methods as per the manufacturer's instructions (Millipore, USA).

The membrane was then washed three times with 0.05% Tween-20/PBS and the reactive bands were visualized with BCIP-NBT solution, as described by the manufacturer.

The blocked membranes were incubated with the appropriate antibody, and the immunoreactive bands were visualized with a chemiluminescent reagent as recommended by Amersham Biosciences, Inc.

The DNA bands were visualized by silver staining and developed as previously described Sanguinetti et al. [ 14].

Membranes were washed five times with TBS and bands were visualized with the p-MEK antibody, as described above.

After washing the blots as previously, bands were visualized by adding luminal substrate to the blots and exposure to the BioMax film (Kodak).

After washing three times in PBS +0.1% Tween, membranes were incubated in the appropriate second antibody for 2 h at room temperature, washed three times as above and bands were visualized by the ECL system (GE Healthcare).

The protein bands were visualized by Luminata Classico Western HRP Substrate as per the manufacturer's instructions (Millipore).

The bands were visualized by an enhanced chemiluminescence detection system, as recommended by the manufacturer (SuperSignal ULTRA; Pierce, Rockford USAA).

Secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (1 2000) were incubated for 1 h at room temperature and then bands were visualized by chemiluminescence ECL (Thermo Scientific, Rockford, IL) as recommended by the manufacturer.