Many autophagy related proteins, like Beclin1, ATG7, etc. have a certain half life time. Which proteins are degraded at a relative high speed (so within 24 hours)? Does anyone tested a certain target (ATG) using western blot or another approach using human primary cells?
The core autophagy machinery is quite stable (Atg5, Atg7, Beclin1) and are not degraded in an autophagy-dependent manner. If you are looking for a readout of autophagy the best and most reliable if p62/SQSTM1. This is a well characterised protein and its stability is absolutely dependent on autophagy. Under amino acid starvation it will disappear in 2-4h almost completely. Using BafiilomycinA1 or Chlorquine will stabilise the protein as they inhibit the lysosome-mediated degradation. Atg8/LC3B is also a good readout for autophagosome formation as it is conjugated directly to the AP membrane (LC3B-II form).
I hope this helps, let me know if you have any other questions.
Indeed, p62/SQSTM1 is a good readout to investigate autophagy activity and autophagy flux, both assays which we established (also using BFLA1, etc.) I was more looking for a protein in the core machinery which could be targeted in human primary cells.
if you want an ATG to assess autophagic activity then ATG5 would be your best candidate. Less ATG5 and more ATG5-ATG12 complex means more autophagic flux. I have no idea about the half-life time however.
Dear Peter, If you look for a protein in the core machinery which expression is required for autophagy in human cells, I suggest VMP1. Ropolo et al, JCB2007; Vaccaro et al, AUTO2008
- Badges
- Science method