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. 2019 Nov 18;8:e48255. doi: 10.7554/eLife.48255

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© 2019, Herring et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A: left panel) Schematic of known murine CLU mRNA transcripts provided in the NCBI database. (A: right panel) Specific CLU gene transcript levels were examined using 25 ng cDNA isolated from DIV 9/16 neurons/astrocytes. Amplicons were visualized on a 4% agarose gel to ensure the appropriate size (left panel) and a comparison of CLU amplicon intensity was visualized (right panel). (B) Primary neurons (left panel) or astrocytes (right panel) were prepared as indicated. At DIV 9/16, cytosolic and nuclear fractions were isolated. 30 μg of each fraction was analyzed for CLU protein expression using anti-CLU H-330 and anti-CLU M-18. Fraction purity was analyzed using Gapdh (cytosol), Grp75 (mitochondria), and lamin A (nucleus). Isolation of cell type was confirmed by double labeling with either MAP2 (neurons) or GFAP (astrocytes) and CLU H-330, as indicated in the Materials and methods. Cells were imaged at 40X (air; neurons) and 60X (oil; astrocytes) using a customized Olympus IX81/spinning disk confocal inverted microscope and analyzed using the Slidebook Software.