In order to identify proteins that bind to p62's UBA domain, p62 UBA pull down assays were performed. Out of the 24 mixed protein pools, several pools contained [ 35 S] methionine-labeled bands in the primary p62 UBA pull down assays (Fig. 4) . We chose 6 pools (pool # 2, 4, 8, 14, 20, 21) because of their stronger signal to specifically identify which individual protein pool in the mixed pools has the ability to bind to p62's UBA domain. Therefore, a secondary screen was conducted on the 6 positive individual mixed pools (representing 24 individual protein pools) which bound with p62's UBA domain (Fig. 5) . Mixed protein pool # 2 generated a positive protein with molecular weight of 51 KDa (Fig. 4) , and only individual protein pool "c" out of the four protein pools (a, b, c, d) that comprised protein pool #2 had a protein with the same molecular weight (Fig. 5) . Depending on the size of the protein pulled down in the secondary screen compared to the primary screen (Fig. 4) , individual protein pools "c", "h", "i", "o", "t", and "v" were identified (Fig. 5) . To specifically identify which protein in the individual protein pool has the ability to bind p62's UBA domain, the cDNAs from the positive individual protein pools were then transformed into JM109 competent cells and plated out on LB ampicillin plates. Individual colonies were chosen to grow overnight in 1 ml of LB media plus ampicillin. Plasmid DNAs were purified from the cell culture and used for TNT Quick coupled in vitro transcription/translation. The individual protein synthesized from each plasmid DNA was retested for its ability to bind p62's UBA domain. By synthesizing individual protein from individual plasmid using the Gold TNT Quick coupled in vitro transcription/translation system and subjecting them to p62 UBA pull-down assays, 11 positive clones were isolated from the 6 positive individual pools. It is not surprising that 5 more clones showed binding ability with p62's UBA domain since there are 100 cDNAs in each positive individual pool and some of them could have lower binding ability and therefore showed weak signal in the mixed protein pool. It is also possible that they are not as efficiently synthesized in the mixed TNT reaction as in the individual TNT reaction in which only one cDNA was used as template. The 11 positive plasmids were sequenced and compared with known cDNA sequences in NCBI database using BLAST analysis with results shown in Table 1 .