Fig. 4: OTUD5 regulates the protein stability of sestrin2, a feedback inhibitor of mTOR.

A The interaction diagram between OTUD5 and specific proteins in the mTOR signaling pathway was obtained by mass spectrometry analysis. B Confocal immunofluorescence microscopic analysis of OTUD5 and sestrin2 in 253 J and UM-UC-14 cells. Scale bars represent 10 μm. C IB analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-OTUD5 and HA-tagged sestrin1-3. pcDNA3.1 was used as the control. D IB analysis of WCL derived from 253 J and UM-UC-14 cells stably expressing shOTUD5 and T24 cells stably overexpressing OTUD5. p-mTOR, p-4EBP1 and sestrin2 were detected, β-actin was used as the loading control. E GST pull-down assay revealed no direct interaction between sestrin2 and OTUD5. The upper panel presents the result of IB using the antibody against HA, and the lower panels show Coomassie blue staining of the purified proteins. F IB analysis of WCL and Ni-NTA pull-down products derived from 293 T cells transfected with Flag-OTUD5 WT, Flag-OTUD5 C224S, HA-sestrin2 and His-Ub. 20 μM MG132 was added 6 hours before harvesting the cells. G OTUD5 knockdown cells (shOTUD5) as well as parental UM-UC-14 cells (shNC) were treated with 100 μg/ml cycloheximide (CHX) for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. H Quantification of the band intensities in G. Sestrin2 levels were normalized to the corresponding β-actin levels, then normalized to the t = 0 h sestrin2 level. I IB analysis of WCL derived from 293 T cells transfected with HA-sestrin2, Flag-EV and Flag-OTUD5. Cells were treated with 100 μg/ml CHX for the indicated time period before harvesting. EV, empty vector. J Quantification of the band intensities in I. Sestrin2 levels were normalized to the corresponding β-actin levels, then normalized to the t = 0 h sestrin2 level. All data are presented as mean ± SD of 3 independent experiments.