Dear all
I want to study the autophagy levels in liver samples of wistar rats under a 20% energy restriction. Among other autophagy "markers", I will tray to measure the ULK1 phosphorylation state by western blot. The problem is that I don´t know which serine phosphorylation should I measure in order to know the autophagy level.
Any advice/suggestion would be very appreciated.
Thanks for reading
Iñaki Milton
Dear Iñaki,
I suggest checking the phosphorylation of ULK1 at S555 for AMPK-dependent phosphorylation and at S638 or S757 for mTOR-dependent phosphorylation. I can see heavy dephosphorylation of S757 after amino acid starvation-induced autophagy as described in Shang et al., 2011.
The antibodies from CST work really fine.
I further suggest checking for LC3, which is a better marker for autophagy. Lower levels of p62 can be indicative of autophagy, too. Further autophagy markers are listed in Klionsky et al., 2016.
Kind regards,
Jana
Alers et al., 2011: https://www.ncbi.nlm.nih.gov/pubmed/22025673
Shang et al., 2011: https://www.ncbi.nlm.nih.gov/pubmed/21383122
Klionsky et al., 2016: https://www.ncbi.nlm.nih.gov/pubmed/?term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition)
Antibodies:
https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser555-d1h4-rabbit-mab/5869
https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser638-d8k9o-rabbit-mab/14205?N=4294956287&Ntt=ulk1+&fromPage=plp
https://www.cellsignal.com/products/primary-antibodies/phospho-ulk1-ser757-d7o6u-rabbit-mab/14202?N=4294956287&Ntt=ulk1+&fromPage=plp
Dear Jana
Thank you very much for your help. I wiil check the papers + antibody references you have attached in your answer. We have already thought in checking the LC3 and p62 levels too.
Thanks again
Iñaki
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