This is obviously protein fragmentation, if you see this band consistently at 1 hour post treatment and can exclude sample mishandling i.e. some samples stayed at room temperature too long or you heated them for too long during WB preparation or anything similar. Than your treatment cause your target protein to fragment. You can clearly see that antibody recognize correct epitope sequence in smaller sized bands. How to discuss or speculate about it depends on protein of interest. If you want to learn more about what it is going on within the cell try to resort to immunofluorescence of protein of interest and see how it's dynamics changes within the cells 1 hour after treatment. If it is something of particular interest to your project can always excise the band from the gel and read amino acid sequence by Mass Spec then you can overlay the fragment sequence to canonical sequence (or excise and read the main band) and learn what part of protein exactly are fragmented and at at which places protein is cut/broken. Hope it helps.

Dear Dr. Anton Lennikov,

Thank you very much for the explanation and suggestion. I do not have any funding for Mass Spec that I wish to do so. But I may try immunofluorescence. I have tried different cell lines and antimycin and oligomycin alone, I did not get the same result. I do not understand why my cell line has such fragmentation.

Probably, I will try MG132 to inhibit proteolytic activity to see fragmentation again. If I have a time and funding, I would like try other mitophagy inducers Pink1/Parkin independent way.

One last question, do you think this can be results of apoptosis? After 4-8hours, cells start to die.

It is certainly can be a sign of apoptosis as proteins in dead cells rapidly degenerate due to lysosomal digestion (protease/phoshpotase inhibitor cocktail is added to be lysis buffer to block this activity). Digested proteins are fragmented (and thus recognized as smaller size bands), but still may have the intact epitope amino acid sequence to be recognized by the antibody.

Probably proteolysis was activated significantly earlier (and indepedently) than apoptosis. Mitochondria contain several proteases that are activated upon dysfunction of the bioenergetics mechanisms.

For review see Article Mitochondrial proteolysis: Its emerging roles in stress responses

Hi Suleyman,

I totally agree with the previous posts.

Also, it's definitely a good idea to go for MG132 as a potent proteasome inhibitor. To further explore which proteolytic enzymes might be responsible for the observed fragmentation, you could conceive broadening your selection of inhibitors - if available to you - which target proteases such as caspases and calpains. The latter ones were, to my knowledge, not mentioned in suggested review.

It bears mentioning that CCCP and the AO treatment appear to induce the proteolytic fragmentation of your protein of interest with a different kinetic / intensity as the AO treatment shows two main fragment bands at 1 h, which disappear after 4 h, but are not present in the CCCP treated samples from the beginning. Thus, this specific fragment might be an intermediate product, which is further cleaved down during the treatment course.

Now some speculation from my perspective: As CCCP, oligomycin and antimycin A may have effects on the mitochondrial / cellular calcium homeostasis, the calcium-dependent calpains may be activated somehow directly by the treatment. So perhaps it's worth going for a calpain inhibitor or a cell-permeable calcium chelator (like BAPTA) treatment to see what happens.

Moreover, you can also use some online-available cleavage prediction tools to check if your protein is a potential substrate for certain proteases.

Are you using protease and phosphatase inhibitors and EDTA in your lysis buffer?

Dear Salah Sommakia ,

I am using homemade RIPA lysis buffer (includes EDTA, Sodium Fluoride, Sodium orthovanadate, PMSF ) and Roche protease inhibitor cocktail I am using.

Dear Anton Lennikov

yes, I am using phosphate and protease inhibitors in my lysis solution. Therefore, it should be another reason for it, right?

Dear Boris V Chernyak

I only knew about PARL. Thank you very much for suggested article. Not for this project but maybe later I may use siRNA or inhibitors of individual enzymes to find out which is the player for this process and wheteher this is important or not.

Dear Jonasz Jeremiasz Weber

Thank you very much for this long and beneficial comment.

First and foremost, I want to tell that I used different cell line more than 5 or 6 different. Only my model cell line and Hek293T shows the similar pattern. But I am not quite sure for the Hek293T because I checked one time. But I am pretty sure about my result. Therefore it should be some cell specific cleavage or some enzyme dependent.

I have not tried prediction tools before It is really useful believe and I used expasy peptidecutter and I showed a lot of cuting enzymes you may find the result in the attachment.

I have recently learnt that proteosome cleavage is very rapid and products dont last long but I am observing the fragment even in 8 hours sample. If I have a funding sometime later, I may try calcium-dependent calpains it really makes sense.

Lastly, you pointed out a good point. I used half concentrations of AO and observed fragments later than 1x AO mix. I used 10 uM CCCP, maybe if I use lesser concentations, I may see the third fragments like in AO treatment.

Additionally, I want to use iron chellattor to induce mitophagy other than PINK/PARKIN pathway to see whether this fragmentation is dependent upon this pathway or not.

Best regards,

Suleyman.