Autophagosome immunoisolation from GFP-LC3B mouse tissue

Abstract

We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4-5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The postnuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis.

Autophagy protein 5 controls flow-dependent endothelial functions

“…LC3-positive membrane-bound vesicles were isolated as previously described by Yao et al [ 68 ]. Briefly, primary lung endothelial cells from GFP-LC3 mice were grown on T75 flasks until confluence.…”

Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function

“…The protocol for autophagosome isolation from H9c2 cells was modified from protocols described in a previous report (Yao et al, 2019 ). In brief, H9c2 cells were first transfected with adenovirus encoding either GFP or GFP-tagged LC3, and then cells encoding GFP-LC3 were treated with either vehicle (Veh) or CQ at 20 µM for 16 h under nutrient-replete conditions.…”