Extended Data Fig. 2: The screening of the DUB expression library.

Experiments were performed as indicated three times with similar results. a–p, CHO-7 cells were set up for experiments on day 0 at 4 × 10⁵ cells per 60-mm dish in DMEM/F12 supplemented with 5% FBS. On day 1, cells were transfected in 3 ml of DMEM/F12 supplemented with 5% FBS containing 1 μg of pCMV-HMGCR-T7, 30 ng of pCMV-Insig-1-Myc and 0.3 μg indicated Dub. The total DNA was adjusted to 2 μg dish^-1 using pcDNA3 mock vector. Eight hours after transfection, cells were incubated in 3 ml of DMEM/F12 supplemented with 5% FBS. On day 2, cells were washed with phosphate-buffered saline (PBS) and then switched to DMEM/F12 containing 5% lipoprotein-deficient serum (LPDS), 1 μM lovastatin, and 10 μM mevalonate. After incubation for 16 h at 37 °C, the cells were treated with 1 μg ml^-1 25-HC plus 10 mM mevalonate as indicated. After 5 h at 37 °C, cells from 2 dishes were pooled and collected, lysed, and subjected to immunoblotting. Immunoblot analysis was carried out with anti-T7 IgG (against HMGCR) and anti-FLAG IgG (against DUBs) as described. Mev., mevalonate.