Fig. 1 | Nature Communications

Fig. 1

From: The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins

Fig. 1

Overview of LipoGlo assays and experimental manipulations. a Individual larvae carrying the apolipoprotein B (ApoB)-NanoLuc reporter are first homogenized in ApoB-containing lipoprotein (ApoB-LP) stabilization buffer. Homogenate can be used for LipoGlo counting (a plate-based assay for NanoLuc activity to measure the total number of ApoB-LPs), LipoGlo electrophoresis (a Native-polyacrylamide gel electrophoresis (N-PAGE) assay to determine the ApoB-LP size/subclass distribution), and DNA extraction for genotyping. Alternatively, lipoprotein density and size can be determined by density gradient ultracentrifugation (DGUC) followed by electron microscopy. To determine localization of ApoB-LPs in situ, individual larvae are fixed in 4% paraformaldehyde (PFA) and mounted in low-melt agarose for chemiluminescent imaging (LipoGlo microscopy). b ApoB protein fused to NanoLuc is loaded with lipid through the activity of microsomal triglyceride transfer protein (Mtp) to form very-low-density lipoprotein (VLDL) particles. In the absence of lipidation, the protein will be rapidly degraded by endoplasmic-reticulum-associated protein degradation (ERAD). VLDL is lipolyzed by serum lipases that use Apoc2 as an obligate cofactor to produce smaller lipoprotein classes such as LDL. Here we investigate the effects of (i) genetic manipulations (mutations in mtp and apoc2), (ii) dietary variation (fasting and feeding), and (iii) pharmacological treatment (inhibition of Mtp with lomitapide) on various aspects of the ApoB-LP profile

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