Figure 9

LMP1-induced sumoylation of SENP2 at K48 and K447 did not contribute to LMP1-mediated modulation of SENP2 stability and localization. HEK 293 cells were transfected as indicated. 48 hours post-transfection, cells were collected and (a) Western blot analyses of SUMO-AMC lysates were 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) treated with cyclohexamide (CHX) or the vehicle control (DMSO) for 18 hours prior to harvesting. Slot blot analyses were performed to detect total SENP2 levels. (d) WCL and NE fractions were collected. Western blot analyses were performed to detect SENP2 (GFP) and LMP1. GAPDH and PARP were used as loading controls. (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.