Fig 1: CLU protein is expressed in the brain as multiple protein isoforms that have distinct cellular localizations.(A) Whole brain, liver, lung, and heart tissues were isolated from age-matched male (left panel) and female (right panel) wild-type (WT) mice and homogenized as indicated. Total protein was analyzed with SDS-PAGE and blots were probed for CLU immunoreactivity with anti-CLU H-330. (B) Cytosolic, organelle, and nuclear fractions were isolated from freshly harvested WT cortical tissue as indicated. Fractions were analyzed via SDS-PAGE and blots were probed for CLU immunoreactivity with anti-CLU H-330 (left panel) and anti-CLU M-18 (right panel). Blots were stripped and re-probed with fraction-/organelle-specific biochemical markers: Grp75 (mitochondria), calnexin and calreticulin (ER), lamin A (nucleus), and Gapdh (cytosol). (C) 40-µM-thick rodent brain sections were permeabilized and blocked as indicated and labeled with anti-MAP2 (green) or anti-GFAP (Figure 1—figure supplement 1C) and anti-CLU H-330 (red). Brain sections were then washed and probed with anti-mouse FITC (for MAP2) or anti-rat Cy5 (for GFAP) and pre-adsorbed anti-rabbit Cy3 (for CLU). To generate a secondary antibody control (bottom panel), one group of free-floating brain sections was incubated overnight in the same conditions without primary antibody. Brain sections from the hippocampal dentate gyrus were imaged at 4X (Figure 1—figure supplement 1B) and 40X using a customized Olympus IX81/spinning disk confocal inverted microscope and analyzed using the Slidebook Software.