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. 2011 Nov 8;287(4):2317–2327. doi: 10.1074/jbc.M111.273730

FIGURE 3.

FIGURE 3.

CFTR-ΔF508 protein aggregates contain unanchored ubiquitin generated by deubiquitinase ataxin-3. A–C, the specificity of the C-terminal ubiquitin anti-UbiC antibody. A and B, AFC-conjugated ubiquitin (lane 1), pure monoubiquitin (lane 2), and tetraubiquitin (lane 3) peptides were resolved by SDS-PAGE and detected in Western analysis by either anti-UbiC antibody (A) or anti-UbiN antibody (B). C, ubiquitin C-terminal truncation mutants Ub71 (lane 1), Ub74 (lane 2), and Ub75 (lane 3) and point mutant G75A/G76A were resolved by SDS-PAGE and detected in Western analysis by either anti-UbiN antibody (top panel) or anti-UbiC antibody (bottom panel). D–H, cultured 293T cells were transfected with GFP-CFTRΔF508 and either scrambled (lanes 1–6) or ataxin-3 siRNA (lanes 7–12) and treated with either Me2SO (lanes 1–3 and 7–9) or MG132 (lanes 4–6 and 10–12). Cell lysates were collected 2 days after transfection. GFP-CFTRΔF508 aggregates were immunoprecipitated with GFP antibody and resolved by SDS-PAGE and probed for GFP (D), ubiquitin C termini by anti-UbiC antibody (E), ubiquitin N termini by anti-UbiN antibody (F), HDAC6 (G), and ataxin-3 (H). IB, immunoblot; Ub, ubiquitin.