Using western blot, LC3-II ( rerpresenting the autophagosomes or the autolysosomes) decreased in treated cells compared to control while using IF, LC3 puncta ( rerpresenting the autophagosomes or the autolysosomes) increased in treated cells compared to puncta. Thanks in advance.
1. what kind of lysis buffer that you used for WB, whether it can break the nuclei membrane. lots of LC3 in nuclear.
2. what antibody are you using, do you know which epitope that the antibody recognizes? not sure LC3 cleavage?
Junjie Shao Thanks for answering.
1. I used Ripa buffer .
2. Anti-LC3B is developed in rabbit using as immunogen a synthetic peptide corresponding to amino acids 2-15 of human LC3B (Gene ID: 81631), conjugated to KLH via a C-terminal cysteine residue and it bind both the cytoplasmic LC3-I and the autophagsomal LC3-II.
Is it possible that the LC3-I are forming aggregates in treated cells so that it looks like more autophagosomes are generated using IF especially that treated cells showed a greater increase in the LC3-I proteins levels using western blot compared to control.
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