FIGURE 8.

Overexpression of SUMO and Ubc9 enhances SUMOylation at K669. The three mutant cell lines (A–C) were transiently transfected with mCherry or mCherry + SUMO + Ubc9. Cell lysates were used in IP experiments with anti-GFP antibodies. WBs containing IP products were probed with the anti-SUMO2/3 antibody, stripped and reprobed with the anti-GFP antibody. Top Panel: Representative blots showing the GFP-HCN2 doublet that was measured. Bottom Panel: Plots of the fraction of SUMOylated GFP-HCN2 channels in each mutant cell line, mean + SEM. The treatment and the n are shown below the graph. Each n represents one plate that was transfected and carried through the experiment to produce a single lane on a WB. Asterisk significantly different (Student’s t-test, p < 0.05).