Figure 2

Identification of the ubiquitination sites on UBXN7. (A) Mass spectrometry data of enzymatic digested peptides from His-UBNX7 was acquired on Nanoflow UPLC and analyzed using MaxQuant. (B) The two ubiquitination sites of His-UBXN7 on K14 and K412 are highlighted and shown on the peptide sequence. Trypsin digestion of ubiquitin conjugates generates a diGly tag that is formed at the ubiquitinated lysine residue. (C) Schematic diagram of UBXN7 protein showing the different domains and indicating the respective ___location of the two ubiquitination sites. The K14 site is located in the UBA ___domain that is responsible for interaction ___domain with HIF-1α, whereas the K412 site is in the UBX ___domain which interacts with AAA + ATPase p97. (D) Amino acid sequence alignment of the two ubiquitin-modified lysine residues to highlight conservation among species. (E) To confirm UBXN7 ubiquitination at the specific lysines, both K14 and K412 were replaced with arginine (R). HEK293 cells were transfected in duplicate with the indicated plasmids: GFP-MUL1, pcDNA-HA-Ub, and His-UBXN7 wild type (wt), single mutants His-UBXN7-K14R or -K412R and double mutants His-UBXN7-K14R-K412R. Cell lysates from control and MG132 treated plates were immunoprecipitated using anti-His antibodies, the precipitate resolved by SDS-PAGE and Western blot analysis using HA antibodies. Lower panel shows His-UBXN7 expression. (F) Western blot analysis of the transfected cells used in (E) to verify overexpression of the different His-UBXN7 constructs does not affect the endogenous level of UBXN7 protein.