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. 2011 Mar 24;7(3):e1001325. doi: 10.1371/journal.ppat.1001325

Figure 3. Cif selectively inhibits USP10 activity in early endosomes.

Figure 3

A. Cif-containing OMV reduced the activity of a 110 kDa deubiquitinating enzyme (DUB) as assessed by a DUB activity assay in cells treated with ΔCif-OMV (Control) or Cif-containing OMV (15 min treatment; see Methods and [20], [21], [45]). The DUB activity assay employs a HA-UbVME probe that forms an irreversible, covalent bond only with active DUBs. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME-DUB complex using an anti-HA monoclonal antibody followed by SDS-PAGE and western blot analysis. The 110 kDa DUB was identified as USP10 by Western blot. USP34 and USP8 were also identified in early endosomes by western blot, however, the DUB activity assay revealed that USP34 was active, but its activity was not altered by Cif. By contrast, USP8 activity was not detected. Quantitation for all western blot experiments is presented to the right. All experiments were repeated at least 3 times, * p<0.05. 95% confidence intervals (USP10: control, 100 to 100; OMV, 61.66 to 39.20; USP34: control 100 to 100; OMV, 136.70 to 44.31). B. siRNA knockdown of USP10 abundance recapitulates the Cif-mediated increase in CFTR ubiquitination, as assessed by immunoprecipitation of CFTR and western blotting for multi-ubiquitinated CFTR in Cif OMV or siRNA-USP10 treated cells. Experiments were performed in the presence of 200 µM chloroquine to allow accumulation and detection of ubiquitinated CFTR. siNeg, nonspecific, scrambled siRNA served as negative control for siRNA transfections. IgG, immunoprecipitation using a non-immune IgG was used as a negative control. Black boxes highlight multi-ubiquitinated CFTR on blots. Quantitation for western blot experiments is presented beside representative blots. All experiments were repeated at least 3 times, * p<0.05. 95% confidence intervals (siNeg, 0.88 to 1.14; siNeg+OMV, 1.87 to 3.30; siUSP10, 2.35 to 3.61). C. siRNA knockdown of USP10 reduces USP10 protein levels in airway cells, as assessed by western blot analysis. A USP10 representative western blot is shown for cell lysates from experiments in Figure 3B, detecting ubiquitinated CFTR.