FIGURE 4.

Transient transfection with SUMO and Ubc9 leads to an increase in GFP-HCN2 channel SUMOylation. The stable Hek-HCN2 cell line was transiently transfected with mCherry (control), or mCherry + SUMO + Ubc9, or mCherry + SENP1. Two days after transfection, cell lysates were used in IP experiments with an anti-GFP antibody. IP products were resolved with SDS-PAGE and transferred to WBs. Blots were probed with an anti-SUMO2/3 antibody. After recording the result, blots were stripped and reprobed with an anti-GFP antibody. (A) Representative blots showing typical chemiluminescent signals for the GFP-HCN2 channel doublet after probing with the anti-SUMO2/3 antibody (upper panel) followed by stripping and re-probing with the anti-GFP antibody (bottom panel). Note that the amount of IP product varied between experiments but not across treatment groups as determined by measures of the GFP OD’s [one-way ANOVA, F(2,16) = 0.1285; p = 0.8804]. (B) The fraction of SUMOylated HCN2 channels in each treatment group (SUMO doublet OD ÷ GFP doublet OD, see text) is plotted as the mean + SEM. The treatment and the n are shown below each plot. Each n represents a single plate that was transfected and carried through the experiment to produce a single lane on a WB. Asterisk represents a statistically significant difference from control [one-way ANOVA with a Dunnett’s post hoc, F(2,16) = 4.121; p = 0.0360].