HDAC6 ZnF-UBP domain interacts with CFTR-ΔF508 aggregates and ubiquitin C terminus. A, protein-protein interaction assay of ubiquitin with either His-tagged wild-type HDAC6 ZnF-UBP domain, a R1155A/Y1184A mutant, or wild-type USP5 UBP domain. His-tagged proteins were incubated with or without ubiquitin at room temperature for 30 min before Ni-NTA was added. The resin was washed extensively prior to elution with SDS-PAGE loading buffer. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. B, both HDAC6 ZnF-UBP and USP5 UBP, but not HDAC6 ZnF-UBP RY mutant, interact with GFP-CFTR-ΔF508 protein aggregates. Cultured 293T cells were transfected with GFP-CFTR-ΔF508 and treated with either MG132 or Me2SO, as indicated. The lysates were incubated with HDAC6 ZnF-UBP (top panel) or HDAC6 ZnF-UBP RY mutant (bottom panel). CFTR-ΔF508 aggregates were immunoprecipitated with an anti-GFP antibody, resolved by SDS-PAGE, and visualized by Coomassie staining. C, both HDAC6 ZnF-UBP and USP5-UBP interact with ataxin-1-82Q-GFP aggregates. Cultured 293T cells were transfected with human ataxin-1-82Q-GFP and treated with either MG132 or Me2SO, as indicated. The lysates were incubated with HDAC6 ZnF-UBP, HDAC6 ZnF-UBP RY mutant, or USP5-UBP. Ataxin-1-82Q-GFP aggregates were immunoprecipitated with an anti-GFP antibody, resolved by SDS-PAGE, and visualized by Coomassie staining. D, GFP overexpression did not cause aggresome formation. The cells were transfected with pLenti-GFP construct and treated with either Me2SO or MG132 in Me2SO. The cell lysates were collected 2 days after transfection; incubated with either USP5 UBP, HDAC6 ZnF-UBP, or HDAC6 ZnF-UBP-RY mutant proteins; and immunoprecipitated by anti-GFP antibody. Input and precipitated samples were resolved by SDS-PAGE and visualized by Coomassie staining. E, protein-protein interaction assays of His-tagged HDAC6 ZnF-UBP domain with ubiquitin and ubiquitin-AFC. The His-tagged HDAC6 ZnF-UBP domain was incubated with ubiquitin or ubiquitin-AFC before applied to a Talon (cobalt) column. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. F, protein-protein interaction assays of N-terminal His-tagged ubiquitin and ubiquitin mutants with HDAC6 ZnF-UBP domain. N-terminal His-tagged ubiquitin and ubiquitin mutant proteins were incubated with HDAC6 ZnF-UBP domain before applied to a Talon (cobalt) column. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. MW, molecular mass.