Abstract：
The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.
摘要：
自噬途径中非经典功能的鉴定和表征揭示了复杂的细胞过程，包括LC3相关的吞噬作用（LAP）和LC3相关的内吞作用（LANDO）。这些现象在ATG8与单膜吞噬体和内体的结合中起着关键作用，阐明自噬和细胞稳态之间的动态相互作用。这里,我们提出了LAP定性和定量评估的详细方案，包括免疫荧光，流式细胞术,和分离的LAPosomes的Western印迹。此外,概述了通过免疫荧光检测受体再循环来评估LANDO的方案.本文提出的方法为寻求解开LAP和LANDO复杂性的研究人员提供了实用指南。通过提供分步说明，伴随着对潜在挑战和优化策略的洞察，本章旨在授权研究者探索自噬蛋白的这些非规范功能。
