U2AF65 protein expression by colon tumor stage. Total protein (25 μg) from cytoplasmic (cyto) and nuclear (nuc) colon tumor and normal tissue extracts were separated using 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Blots were incubated with anti-U2AF65 antibody MC-3 and detected using chemiluminescence and autoradiography. Blots were reprobed with an anti-actin antibody, and densitometry was performed using NIH Image J software. U2AF65 expression values were normalized by dividing the actin expression values in each extract, and plotted according to colon tumor stage using the R program (Additional file6).