LC3-II is localized to the isolation membrane (phagophore) and the autophagosomal membrane.Induction of autophagy cannot be determined by simply detecting an increase in LC3-II band intensity on Western blotting.
The Autophagy Flux Assay compares samples treated with or without lysosomal inhibitors to allow assessment of the induction of autophagy.
Reference ：Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 3, 542-5 (2007) (PMID: 17611390)
|Anti-LC3 mAb-HRP-DirecT||8E10||Mo IgG2aκ||100 µL||WB||Hu, Mo, Rat, Hm|
|Anti-LC3 mAb||4E12||Mo IgG1κ||50 µL, 2mg/mL||WB(weak), IC, IP, FCM, Immuno-EM||Hu, Mo, Rat, Hm|
|Anti-α-Tubulin pAb-HRP-DirecT||Polyclonal||Rab IgG(aff.)||100 µL||WB Positive Control||Hu, Mo, Rat, Hm,Chi|
|Positive control for anti-LC3 antibody||100 µL (20 tests)|
|Chloroquine solution (x1000)||100 µL|
|Bafilomycin A1 solution (x1000)||100 µL|
|Cell lysis buffer (x5)||1 mL x2|
Application WB: Western Blotting, IH: Immunohistochemistry, IC: Immunocytochemistry, IP: Immunoprecipitation, FCM: Flow Cytometry
Species Cross-Reactivity Hu: Human, Mo: Mouse, Rab: Rabbit, Hm: Hamster, Chi: Chicken
LC3-II is increased in cells under starvation conditions, compared with cells under control (nutrient) conditions (Lanes 1, 2). When starved cells were treated with the lysosomal inhibitor chloroquine or bafilomycin A1, LC3-II band intensity is further increased (Lanes 3, 4). This increase indicates an accumulation of autophagosomes caused by the inhibition of their degradation. Induction of autophagy in starved cells can be confirmed by comparing these results.
Autophagosomes can be seen as punctate staining inside the cells starved in HBSS (Hank's Balanced Salt Solution). The addition of the inhibitors increases the number of autophagosomes.