Hi Nirmala,

you should take a look at the sequence of your shRNA and align it to the LC3I and LC3II gene, probably the shRNA is more sensible to the second and therefore acts the way it's done.

fred

Hi Frederic,

I really appreciate your suggestion and thank you so much for quick response.

If LC3I and LC3II proteins are induced separately by transcription then I can check the shRNA sequence with both LC3I and LC3II genes as suggested here. However, LC3II protein is the lipidated form of LC3I protein. This means, LC3B mRNA is translated into LC3I protein first and LC3I protein undergoes some modification (lipidation) by which phosphatidyl ethanolamine (PE) is attached to LC3I protein leading to LC3I-PE which is called LC3II protein.

So, LC3II protein is formed from LC3I protein. Therefore, LC3II protein is reduced only when LC3I protein is reduced. However, during autophagy process, sometimes LC3I protein level is reduced but LC3II protein is increased which suggests that LC3I protein is converted into LC3II protein (which is the indication of activation of autophagy not induction of autophagy).

So, in my case also, shRNA should reduce LC3I protein first and then due to the reduction of LC3I protein, LC3II protein should be reduced automatically.

It seems a bit complicated. If you come across any idea after my message, then please keep updating.

Thanks a lot once again Frederic.

Nirmala

Hi Nirmala,

not complicated, just interesting.

does the shRNA interferes with post modification of the LC3I, it seems so. but how? is the modification complex inhibited by the shRNA?

well, you were talking of western blot, do the lipids inhibit antibody capture?

fred

maybe juliano could be some help to you:

https://www.researchgate.net/post/What_is_the_correct_manner_to_represent_the_effects_of_a_treatment_in_autophagic_flux_assay_in_vitro2?_ec=topicPostOverviewQuestionMatchingExpertise&_sg=OL3fSdLJkZ8pkS4zTlgaIKibaEpgUlCbaCivNGkK_itLJkPXdG8IglA2sVvu7JhvTGjecxnnVC77EH_q.Y6qEIQ8Mde0TjDOMdXPCjF02HYLupjQuc4Y7VyUQUxlK22OzagrJPRf7Ep0SHtK5fa2Q6QyQ9ykf5f4PZi1E5yU

fred

Dear Frederic,

The complex of Atg5, Atg12 and Atg16 (other autophagy related proteins) is involved in the process of conversion of LC3I into LC3II protein. So, as you mentioned LC3B shRNA may affect any one of the above autophagy related proteins expression that is why LC3II may be reduced. But I am not expert in checking gene align. So, at this stage it is hard form me to do this.

Further, if may be possible that lipidation may affect antibody capture in Western blot. So, I think I would measure LC3B mRNA expression by RT-PCR and lets see the result.

I will read through the link you have given.

Thank you so much Frederic.

Hi Nirmala

ok, but just keep in mind that sometimes expression of the mRNA does not follow well the amount of protein (lot of regulation process and lifetime of the protein).

fred

Yes, you are right Frederic.

Measurement of mRNA expression may not be reliable in this case. Because if mRNA is effective then LC3I protein is obviously inhibited.

Thank you Frederic.