FIGURE 1.
p97 inhibition reduces the degradation of USP33. A, efficient doxycycline (dox)-induced knockdown of p97 in HeLa p97-V1 cells. HeLa cells were transduced with retrovirus encoding doxycycline-induced shRNA against p97 and treated with the presence or absence of 1 μg/ml doxycycline for the indicated times. Total cell lysates were isolated and analyzed by immunoblotting. B, SILAC data comparing p97-depleted cells with control cells. SILAC-labeled HeLa p97-V1 cells were treated in the presence (heavy-labeled) or absence (light-labeled) of 1 μg/ml doxycycline for 2 days to knock down p97. A mitochondria-enriched membrane fraction was analyzed by mass spectrometry. The SILAC (heavy/light) ratios combined from two independent biological samples are presented. For each indicated protein, at least 10 independent peptide measurements were obtained. Error bars indicate the S.E. Asterisks indicate p value <0.0001. C, analysis of USP33 levels after p97 knockdown. HeLa p97-V1 cells were cultured in the absence or presence of 1 μg/ml doxycycline (dox) for the indicated time, and total cell lysates were analyzed by immunoblotting against USP33, p97, and the loading control Oxa1. D, same as in C, except the murine fibroblast NIH-3T3 and human embryonic kidney 293T cells were used, respectively. E, same as C, except that an independent shRNA (p97-V4) against p97 was used. For control, a nontargeting shRNA was used. F, rescue of p97 knockdown cells. HeLa p97-V1 cells expressing empty vector or an shRNA-resistant version of p97 (p97R) were treated with 1 μg/ml doxycycline to induce knockdown of endogenous p97. Total cell lysates were isolated and analyzed by immunoblotting. G, effects of p97 knockdown on exogenously expressed USP33. p97-V1 cells in which the endogenous expression of USP33 was constitutively silenced (USP33-ShA) were transduced with a retrovirus encoding a shRNA-resistant version of untagged USP33 (exo USP33) where indicated. Cells were treated with or without 1 μg/ml doxycycline to knock down p97 for the indicated time, and the levels of USP33 were analyzed by immunoblotting. H, analysis of USP33 degradation upon chemical inhibition of p97. HeLa cells were treated with 100 μg/ml cycloheximide (CHX) and 10 μm NMS-873 where indicated, and total cell lysates were analyzed by immunoblotting. I, accumulation of ubiquitinated USP33 upon inhibition of p97. HeLa cells were treated with control (dimethyl sulfoxide) or 10 μm NMS-873 for 12 h, and total cellular ubiquitinated proteins were affinity-purified with TUBE2-agarose. Ubiquitinated USP33 (Ub-USP33) was detected by immunoblotting using anti-USP33 antibody.