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. 2021 Jun 1;11:576326. doi: 10.3389/fonc.2021.576326

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Copyright © 2021 Zembroski, Andolino, Buhman and Teegarden

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Validation of cytoplasmic lipid droplet (CLD) isolation. (A) Triacylglycerol (TAG) to protein ratio of each isolated fraction. CLDs were isolated from MCF10CA1a cells using sucrose density gradient ultracentrifugation. Fractions were removed sequentially from the top of the gradient to the bottom. Floating fraction (FF): isolated CLDs, 1-10: soluble fractions, P: pellet. (B) Western blot of isolated fractions and whole cell lysate (WCL). Fractions were loaded by volume: 10 μL FF-10, 5 μL P and WCL. Membrane was probed for markers of CLDs (PLIN3), cytosol (GAPDH), and endoplasmic reticulum (CANX). Approximate molecular weight markers for each protein are listed. See Supplementary Figure 1 for a representative Ponceau stain reflecting the relative levels of protein in each fraction.