Identification of polyubiquitinated proteins binding to UBA domain of hFAF1.

<p>(A) Schematic diagram of UBA domain specific tandem immunoprecipitation of hFAF1. HEK293T cells were co-transfected with Flag-hFAF1 and HA-ubiquitin (or HA-vector). Control cells were co-transfected with Flag-hFAF1 and HA vector, and examined cells were co-transfected with Flag-hFAF1 and HA-ubiquitin. In the first immunoprecipitation, cell lystes were immunoprecipitated with anti-Flag M2 agarose-linked affinity beads. In these precipitates, hFAF1 interactomes via various domains (UBL1 and UBX) were appeared as well as polyubiquitinated proteins bound to UBA domain. Precipitates were eluted by 1% SDS elution buffer and 10x diluted. Second immunoprecipitation with HA affinity beads was performed at room temperature with diluted supernatant. In this IP, only ubiquitinated proteins bound to hFAF1 were precipitated. (B) Second immunoprecipitation with anti-HA antibody was confirmed by Western analysis using anti-ubiquitin antibody. (C) Immunoprecipitated samples were separated in 10% SDS PAGE, and gel silver stained. High molecular weight ubiquitinated proteins binding to hFAF1 were analyzed using UPLC-ESI-q-TOF tandem MS. (D) Hsp70 detected in high molecular weight bands (2–4) were identified by MS/MS as shown by the spectra with K-48 linked ubiquitin chain.