Each translated pool was resuspended in binding buffer (25 mM Tris pH 7.5, 125 mM NaCl, 0.1% NP-40) and used as a source of protein in p62 UBA pull down assays. Proteins that specifically interact with the UBA domain of p62 were isolated by interaction with agarose-immobilised p62-UBA peptide (amino acid 387-436 of p62) (5 µg) for 2 hours at 4ºC, then washed three times in washing buffer (25 mM Tris pH 7.6, 100 mM NaCl, 1% NP-40). Bound proteins were released by addition of SDS-sample buffer and separated by SDS-PAGE. The SDS-PAGE gels were fixed in 50% methanol, 10% acetic acid for 30 min, stained in 0.2% Commassie Brilliant Blue R-250, 45% methanol, 10% acetic acid for 15 min, destained in 10% acetic acid, 50% methanol overnight, and enhanced in autoradiography enhancer En 3 HANCE for 1 hr and exposed to X-Ray film.