It has been reported that polyubiquitin chains assembled through lysine 48 of ubiquitin act as a signal for substrate proteolysis by the 26S proteasome (42) (43) (44) . In order to understand whether the proteins identified in our screen bind to the p62's UBA domain through lysine 48 (K48), polyubiquitin K48 chains were added to the p62 UBA pull down assay (Fig. 6) . Inclusion of polyubiquitin K48 chains in the assay should compete for the binding of substrate to the p62's UBA domain and reduce the interaction of those proteins with the p62's UBA domain if those proteins are assembled through K48 chains. An alternative interpretation for polyubiquitin K48 chain competition is that the ubiquitin chains are competing for the same binding site as the binding partners which are either ubiquitinated or non-ubiquitinated. We randomly chose five proteins out of the 11 binding partners for the competition pull down (Fig. 6) . Out of the five proteins, four proteins (# 2, 3, 4, and 5) showed reduced binding ability with p62's UBA domain when polyubiquitin K48 chains were included (Fig. 6A, 6B ). However, K48 chains failed to compete with HSP70, suggesting that p62's UBA domain binds to HSP70 through a ubiquitin lysine linkage other than K48. Interestingly, it has been reported that heat shock protein 70 cognate (HSP70) is ubiquitinated by CHIP (carboxyl terminus of Hsc70-interacting protein) via ubiquitin chain synthesis that uses either K29 or K63 (45) . In order to examine which lysine linkage utilized by HSP70 binds to p62's UBA domain, in vitro ubiquitination assay was performed by incubating lysates from HEK cells expressing HSP70 with E1, E2, and E3 in reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP). As control, the ubiquitination of HSP70 utilizing the rabbit reticulocyte lysate was also investigated by Western blot analysis. Our results revealed that HSP70 was ubiquitinated in the IVEC system (Fig.  7A, 7B) , and the rabbit reticulocyte lysate contained enzymes such as TRAF6 (E3) and UbcH7 (E2) for in vitro ubiquitination (Fig. 7C ). TRAF6 was chosen as an E3 in this in vitro ubiquitination assay due to its RING domain, a common feature of E3 ligases, and the observation that p62 is a scaffold for TRAF6 interaction (46) . Therefore, in vitro ubiquitination assays using the E1-E2-E3 system were performed in the presence of either ubiquitin wild type or ubiquitin mutants (K29R, K48R, and K63R). If one lysine mutant blocks the ubiquitination of HSP70, it would suggest that the ubiquitination of HSP70 utilizes that specific lysine linkage.