Hie everyone
The treatment used in my study is showing increase in Beclin-1 expression and Lc3b-II turnover in M.bovis infected macrophages. The same treatment led to reduced expression of p62 protein and increased expression of Rab7 in infected macrophages. Are these markers indicating autophagy in my experiments? Do i need to check any other marker ? I also found increased colocalization of M.bovis GFP and Lysotracker upon same treatment through confocal microscopy.
To rule out the influence of autophagic flux, you should perform the experiment using bafilomycinA1 or CQ.
As to the autophagy marker, LC-3 conversion and p62 consumption are enough for you to evaluate the autophagy activity.
Thanks for the reply Dr Yoshida
I tried using 100nM Wortmannin with the treatment and it reversed all the effects on the protein markers.
Do I still need to perform experiments with Bafilomycin A1?
As compared with 3-Methyladenine and Wortmannin which inhibit class III PI3 kinase, Bafilomycin A1 blocks the formation of autophagolysosomes mediated by V-type-ATPase.
In conclusion, you need not perform additional experiment with Bafilomycin A1 if you have already done the experiment with Wortmannin, in which you can rule out the effect of autophagy flux.
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