Research Paper Volume 14, Issue 21 pp 8645—8660

Figure 5. USP7 regulates cyclin F protein stability. (A) HCT116 cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT or empty vector. Twenty-four hours post transfection, cells were treated with DMSO or cycloheximide (CHX; 50 μg/mL) for the indicated minutes, lysed, and immunoblotted as indicated. α-Tubulin was the loading control. (B) HeLa cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT or empty vector. Forty-eight hours post transfection, cells were treated with CHX (50 μg/mL) for 75 min. Where indicated, P22077 (25 μM) was added to cells 2 h prior to the addition of CHX. Cells were then lysed, and immunoblotted as indicated. (C) HeLa cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT, Flag-USP7 CS, or empty vector. Forty-eight hours post transfection, cells were treated with CHX (50 μg/mL) for 75 min, lysed, and immunoblotted as indicated.