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. 2014 May 22;289(28):19789–19798. doi: 10.1074/jbc.M114.569392

FIGURE 5.

FIGURE 5.

HERC2 is required for accumulation of ubiquitinated USP33 upon p97 inhibition. A, analysis of ubiquitinated USP33 in p97 knockdown cells lacking HERC2. HeLa p97-V1 cells were transduced with retrovirus expressing either a control (nontargeting) shRNA or an shRNA against HERC2 (HERC-ShB). Cells were treated with 1 μg/ml doxycycline (dox) for 48 h where indicated, and total cell lysates were isolated. Total ubiquitinated proteins were affinity-purified using TUBE2-resin, and ubiquitinated USP33 (Ub-USP33) was detected by immunoblotting. B, analysis of ubiquitinated USP33 upon NMS-873 treatment in cells depleted for HERC2. Control (expressing nontargeting shRNA) or stable knockdown cell lines of HERC2 (HERC2-ShB) or USP33 (USP33-ShA) were treated with 10 μm NMS-873 for 12 h. Total cell lysates were isolated, and TUBE2-resin was used to affinity purify total ubiquitinated proteins. Ubiquitinated USP33 (Ub-USP33) was detected by immunoblotting.