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. 2017 Jun 7;11:45. doi: 10.3389/fnana.2017.00045

Figure 1.

Figure 1

Validation of sortilin antibodies using sortilin knockout (−/−) and wildtype (+/+) mouse brains, with a characterization of the normal expression pattern of sortilin in rodent cerebrum. Panel (A) is a schematic drawing of the human sortilin protein, with the extracellular, transmembrane and intracellular domains of varying lengths of amino acid (a.a.) residues as marked. The immunogenic synthetic peptide sequences for the extracellular and intracellular C-terminal domain antibodies are also provided. Panels (B,C) show representative immunoblot and immunolabeling results obtained with the goat antibody (Gt Ab) against the extracellular domain in sortilin +/+ and −/− brains. Panels (D,E) show the results obtained with the rabbit antibody (Rt Ab) against the intracellular C-terminal. Both antibodies label a ~100 kDa band in sortilin +/+, but not in −/−, lysates (B,D). Several non-specific bands are visible in the immunoblot with the goat antibody, equally present in the sortilin +/+ and −/− lysates, by extending the time of film exposure (B). With overexposure, two light bands at ~40 and ~15 kDa (pointed by arrow) are also noticeable in the immunoblot of sortilin +/+ lysates with the rabbit antibody (D). Light microscopic images (C,E) show neuronal profiles in the subiculum (Sub) to CA1 transitional region labeled by both antibodies in sortilin +/+, but not in −/−, brain sections. Confocal immunofluorescent images show completely colocalized labeling by the two antibodies in cortical pyramidal-like neurons (F–I), CA3 pyramidal neurons and granule cells of the dentate gyrus (DG) (J–M) in C57BL mouse brain, with granular elements seen intracellularly (I,M). Western blot applications: 12% SDS-PAGE gel and 13 μg equal amount protein loading. Additional abbreviations: CC, corpus callosum; s.p., stratum pyramidale; GCL, granule cell layer; PC, Parietal cortex; I-III, cortical layers; GAPHD, glyceraldehyde-3-phosphate dehydrogenase. Scale bar = 200 μm in (C) applying to (E); 100 μm in (F) applying to (G,H,J–L), equivalent to 25 μm for (I,M).