Figure 5. siRNA knockdown of G3BP1 eliminates Cif inhibition of USP10.

A. siRNA knockdown of G3BP1 prevented the Cif-mediated inhibition of USP10 deubiquitinating enzyme activity in the EE fraction, as assessed with the DUB activity assay (using the HA-UbVME probe). Control cells were treated with Δcif-OMV. Quantification of USP10 DUB activity is presented below. Experiments were repeated at least 3 times, * P<0.05 versus control. 95% confidence intervals (siNeg: Control, 100 to 100; OMV, 61.12 to 34.87; siG3BP1: Control, 100 to 100; OMV, 107.8 to 98.69). B. siRNA G3BP1 prevented the Cif-mediated increase in CFTR multi-ubiquitination, as assessed by immunoprecipitation of CFTR followed by western blotting for ubiquitin (clone FK2 antibody). Experiments were performed in the presence of 200 µM chloroquine to block the degradation of CFTR to allow detection of ubiquitinated CFTR. Black boxes highlight multi-ubiquitinated CFTR on blots. IgG, immunoprecipitation using a non-immune IgG was used as a negative control. Quantification for all western blot experiments is presented at the bottom of the panel. Experiments were repeated at least 3 times, * P<0.05 versus control. 95% confidence intervals (siNeg: Control, 102.6 to 98.30; OMV, 173.9 to 272.4; siG3BP1, Control, 108.2 to 68.72; OMV 96.27 to 51.79). C. siRNA G3BP1 prevented the Cif-mediated lysosomal degradation of CFTR, as assessed by western blot analysis. A similar result was observed for apical membrane expression of CFTR (data not shown). Quantification for all western blot experiments is presented below representative blots. Experiments were repeated at least 3 times, * P<0.05 versus control. 95% confidence intervals (siNeg: Control, 100 to 100; 15 min, 147.1 to 57.14; 30 min, 89.36 to 80.44; 60 min, 73.27 to 39.17; 90 min, 60.72 to 27.02; siG3BP1: Control, 100 to 100; 15 min, 159.4 to 51.42; 30 min, 60.22 to 232.5; 60 min, 110.8 to 334.9; 90 min, 86.24 to 303.4).