Figure 2.

GFP-LC3 is present diffusely in the cytoplasm until it is incorporated into the developing autophagosomal membrane. The accumulation of GFP-LC3 on these structures can be visualized by fluorescence microscopy as bright fluorescent puncta. Fusion with the lysosome can be prevented with chloroquine (CQ) or Bafilomycin A1. Selective disruption of autophagy can be accomplished by using a point mutant of Atg5 in which the acceptor lysine is mutated to arginine (Atg5K130R). This prevents the incorporation of Atg12 and stalls the process before incorporation of LC3, phagophore enlargement, or target engulfment.