Figure 1: PHB and phospho-PHB (pT258) colocalize with AKT and phospho-AKT in the mitochondrial sheath of mouse mature sperm. Colocalization of (a) PHB (magenta) or (b) phospho-PHB (pT258; magenta) with AKT (green) or phospho-AKT (pT308, pS473; green), and the mitochondrial sheath (red) of mouse cauda epididymal sperm, was determined using a mitochondrial membrane potential probe (MITO) and confocal fluorescence microscopy. The inserts showed the image amplified for “Line Scan” colocalization analysis. The plot profiles of MITO (red), PHB (magenta) or phospho-PHB (pT258; magenta), AKT (green) or phospho-AKT (pT308, pS473; green) colocalization along the region of interest (ROI) were constructed and analyzed using LAS AF Lite software, as shown in the last column on the right. The negative controls (the last rows of a and b) were run with rabbit (R-IgG) and mouse (M-IgG) IgG isotypes, instead of specific primary antibodies. Scale bars = 20 μm. For amplified image (insert), scale bars = 5 μm. PHB: prohibitin; AKT: protein kinase B; DAPI: 4',6-diamidino-2-phenylindole.