Hyperphosphorylation of AUF1 in NPM-ALK-expressing cells. Proteins were extracted from pcDNA3 and NPM-ALK-transfected NIH3T3 cells, and 700 μg were immunoprecipitated with the monoclonal anti-AUF1 antibody. The IPs were subjected to 2D gel analysis; after transfer, the nitrocellulose membrane was incubated with anti-AUF1 antibody (time of exposure, 1 minute), carefully stripped, and hybridized with secondary peroxidase-conjugated goat antimouse antibody (GAM PO); no signal was observed after 15 minutes of exposure, indicating that no more antibodies were bound to the membrane (data not shown). The membrane was then stripped again and probed with antiphosphotyrosine (anti-pY) antibody (time of exposure, 2 minutes). The hyperphosphorylated AUF1 variants differentially expressed between control and NPM-ALK-expressing NIH3T3 cells are shown by arrows.