FIGURE 4.

NHE5 and AMPKα2 colocalize in transfected AP-1 cells. A, immunofluorescence confocal microscopy images of AP-1 cells transiently co-transfected (48 h) with monomeric cherry fluorescent protein-tagged NHE5 (NHE5_ChFP, red) and enhanced green fluorescent protein-tagged AMPKα2 (AMPKα2_GFP, green). Overlapping signals in merged image are yellow. B, fluorescence (or Forster) FRET analysis of NHE5_ChFP and AMPKα2_GFP interactions were determined by the acceptor photobleaching method (see ”Experimental Procedures“). Photobleaching of NHE5_ChFP resulted in an increase in the GFP signal previously transferred to ChFP during FRET (left panel). Percent change in average fluorescence per pixel, relative to pre-bleaching of NHE5_ChFP is shown in the right panel. Data are representative of at least three independent experiments. Asterisks indicate p < 0.05, Student's paired t test.