Figure 11

Inhibition of ubiquitination processes mediated the observed changes in SENP2 biology. HEK 293 cells were transfected as indicated and treated with PYR-41 or DMSO for 18 hours prior to harvesting. (a) SUMO-AMC lysates were collected and Western blot analyses performed to determine SENP2 (GFP), LMP1, and GAPDH levels. (b) SUMO-AMC assays were performed, and results are shown as mean ± standard deviation of samples done in triplicate and experiments performed in triplicate. (c) Cells treated with cyclohexamide (CHX) or the vehicle control (DMSO) for 18 hours prior to harvesting. Western blot analyses were performed to detect total SENP2 levels. (d,e) Confocal microscopy was performed at 60X magnification using the Nikon A1 laser confocal microscope. Representative images of the middle slice of the nucleus are shown. (e) Representative image showing LMP1-expressing and non-LMP1-expressing cells that were treated with PYR-41.