i treated apoptosis inducing DRUG in cancer cells in an increasing order, the expression of AKT and p-Akt is found decreasing, How can I explain the DATA ?
TIA
Please upload an image of your WB including the loading control, this will make it easier to interpret your results.
I agree with Sabine, also include the treatment conditions, that way we can be of help. From what I can gather from your post is that you might have less cells thus less protein loaded into the wells but if the loading is the same, then from your treatment conditions might be the answer to that.
Your GAPDH is fine but in case of P-AKT and AKT, the last two lanes appears shady. Your control lanes are also partially shady. Width of the bands are nearly same. It is little difficult to compare with control. So, I would suggest you to repeat your western with lower exposure. After that, if you find AKT and p-AKT to decrease, it might be due to reduction of oncogenic signaling leading to reduced transcription or increased degradation of AKT. Your drug might be affecting molecules involved in these processes. For eg, beta-catenin of Wnt pathway increases AKT transcription. Your drug might be inhibiting Wnt pathway. Since AKT is reduced, p-AKT would obviously reduce.
I agree with Moumita Sarkar, repeat your Western blot with lower amounts of protein and/or lower the exposure time; even your GAPDH is so saturated that one cannot judge the equal/unequal loading of the proteins.
It might be due to improper exposure. Your GAPDH blot is not shady but saturated as suggested by Dr. Strehl. Good luck !!
If you replicate succesfully and the same trend is confirmed:
-total / available Akt is being reduced with its phosphorylation unaffected.
-treatment does not reduce Akt phosphorylation and may -at the low doses - slightly increase it (for this you need less exposure as suggested.)
however even overexpossed as it is the suggestion is inhibition of expression or increased degradation of all Akt prior to its subsequent phosphorylation which is maintained.
I repeated the experiment and the results were same, I watched that the exposure had no error, the same trend followed. The p-akt is expressed higher in the midway of the drug concentration, however the cytotoxicity test showed dose dependent manner Howcan I confirm the degradation of Akt is occurring or some other phenomenon? - Thanks a lot Yannis, Moumita, Sabine.
If there is reduced transcription of Akt,you can check its mRNA level by RT-PCR or qRT-PCR. For degradation case, you can use proteasome degradation blocker MG132 or other proteasome inhibitors along with your treatment. If you find no reduction in AKT by your drug in the presence of MG132, its going for degradation. Al d best!!
NO, protease and proteasome inhibitors are not the same!
Protease inhibitors
A broad-spectrum protease inhibitors and protease inhibitor cocktails have been developed to help protect the integrity of proteins during protein extraction and purification.
Proteasome inhibitors
are drugs that block the action of the proteasome pathway that breaks down proteins.
A simple google search would have answered your question. I recommend that you read some basic text books and/or discuss your experiments with your supervisor.
no it is specifically a blocker of proteasome as said above..good luck
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