FRET between native Kv2.1 and SUMO2/3 in hippocampal neurons. Cultured rat hippocampal neurons were studied by immunostaining and confocal microscopy or acceptor photobleaching, antibody-mediated FRET. Bars in all panels are 10 µm. (A) Wide field photomicrograph showing Kv2.1 in clusters and outside the domains. (a) Dense colocalization of Kv2.1 and SUMO2/3 by thin-section microscopy (0.48-µm slices). (Left) Kv2.1 (green) and nuclear (blue) stain. (Middle) SUMO2/3. Right: Colocalization of Kv2.1 and SUMO2/3. (b) Kv2.1 and SUMO1 imaged, processed, and colocalized as in panel a. (B) Acceptor photobleaching showing FRET between Kv2.1 and SUMO2/3 in 2.48-µm sections. (Left panels) Kv2.1 visualized with Alexa Fluor 488 (green); SUMO2/3 visualized with Alexa Fluor 594 (red); and BLEACH shows the region where Alexa Fluor 594 was photobleached (white line). FRET efficiency calculated for pixels within the bleached area. (Right) Overlay of Kv2.1 and FRET reveals Kv2.1 with SUMO2/3 within (open arrow), at the periphery (solid arrow), and at the cell surface distant from Kv2.1 clusters (arrowhead). Inset is a histogram of the distribution of FRET efficiency for pixels in the bleached region. Pixels with an efficiency <5% are not shown. (C) Acceptor photobleaching demonstrating the absence of FRET between Kv2.1 and GAPDH, a cytosolic protein. Images were obtained, analyzed, and presented as in B.