Figure 1. UBQLN1 interacts non-preferentially with diverse ubiquitin linkages through its UBA domain.

(A) Schematic of UBQLN1^WT and engineered domain deletion constructs missing individual domains. (B,C), 293T cells were transfected with empty vector (EV), FLAG-UBQLN1^WT, domain deletion constructs of UBQLN1 and HA-Ubiquitin^WT. 48 hours post transfection, cells were lysed and Western Blot analyses were performed with the indicated antibody. Where indicated, immunoprecipitation with anti-FLAG conjugated agarose beads was performed. UBQLN1^WT interacts with ubiquitin but UBQLN1^112X and UBQLN1^ΔUBA (both missing UBA domain) lose this interaction. Therefore, UBQLN1 interacts with ubiquitin through the UBA domain. (D) 293T cells were transfected with FLAG-UBQLN1^WT and constructs of HA-Ubiquitin^WT that have all Lysines (K) mutated to Arginine (R) except one for example, HA-Ubiquitin^K33 has all K’s are mutated to R except K33 and therefore overexpressing and promoting formation of K33 ubiquitin chains on substrates. K0 indicates all K’s have been mutated to R and this ubiquitin molecule is can only conjugate to the substrate (mono-ubiquitination) and is incapable for forming ubiquitin linkages. UBQLN1 interacts with WT, K33-, K48-, K63- poly-ubiquitin chains and K0 mono-ubiquitinated substrates non-selectively.