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. 2021 Jan;1865(1):129754. doi: 10.1016/j.bbagen.2020.129754

Fig. 2.

Fig. 2

SCF(Fbxo7) ubiquitinates in vivo both isoforms of UXT and promotes proteasomal degradation of UXT—V2. A) UXT-V2 levels in cellular extracts obtained by cell lysis with NP-40 or RIPA buffer. HEK293T cells were transfected with indicated plasmids and the pellets were lysed with NP-40 or RIPA buffer. Total protein lysates (40 μg) were loaded in gel and the levels of UXT-V2 co-expressed with Fbxo7 or Fbxo7-ΔF-box were analyzed. This blot is representative of a triplicate experiment. B) HEK293T cells were transfected with the indicated plasmids, and the total cell lysates were immunoprecipitated with anti-HA. The HA peptide-eluted fractions were resolved by SDS-PAGE and used for western blot analyses with the indicated antibodies. The ratio between densitometry of the each smear and the corresponding anti-HA band is indicated. C) U2OS cells were transfected with Fbxo7 or Fbxo7-ΔF-box in combination with UXT-V2 or UXT-V1- M13G (D) and treated (2 or 4 h) with cycloheximide (CHX) for indicated times. Protein extracts were separated by SDS-PAGE, and the immunoblots were probed with the indicated antibodies. The quantitative analysis of the bands was performed by Image J and is shown in the graph. E) Similarly, CHX chase assay was performed in the presence or absence of the proteasome inhibitor MG132. The graph below shows the densitometric analysis by ImageJ of each protein band revealed by the indicated antibodies. GraphPad Prism was used to statistical analysis 2way ANOVA (Bonferroni posttest) Assays were performed in biological triplicate. *: p ≤ 0.05 and ** p ≤ 0.01. F) Purified DUBs were used to map polyubiquitinated UXT-V2 purified from HEK293T cells. The nonspecific DUBs (USP21 and vOTU) cleaved all ubiquitin chains; OTUB1 (K48-specific) partially removed ubiquitin chains and OTUD1 (preference for K63 linkages) showed concentration-dependent activity against the substrate. G) A fraction of the eluted proteins from A was subjected to western blot analysis with anti-K48 or anti-K63 specific antibodies. The ratios indicate the densitometry of each smear relative to the anti-HA band for a single experiment.