I have never used your specific inhibitor but I have used Bisindolylmaleimide I , which is a pan PKC inhibitor. I measured activation of phosphorylated and total MARCKS, specific substrate for PKC, to ensure that the concentration used was sufficient for inhibiting PKC activity. I used Bis I at the 5 uM concentration.

What concentration of your inhibitor are you using? How long do you put it on your cells? What are you measuring to ensure that PKC beta is truly inhibited? How are you measuring "down-modulation"? Can you describe in more detail what this means? Maybe adding some data would help for interpretation.

Dear Anne,

first of all thank you for the answer.

However, I used Bisindolylmaleimide I too (1 to 5 uM). It works very well.

The point of my question is different by the way: my inhibitor seems to degrade PKCs  as well as it blocks their activity. I use it at 1-5uM and I made time course analyses starting from 1h to 24h. The decrease of the proteins starts after 2-3h from the treatment. What I ask is if anyone knows why this particular inhibitor has the effect to make PKC levels drop (I am talking about the total amount, not the phosphorylated isoforms of PKC).

Do you have any guess about that?

Thanks again

I understood that you seem to find your PKC protein degraded. I wanted to present my background of PKC inhibitor use before trying to attempt to answer your question. When you detect the protein levels, do you detect lysates treated with and without inhibitor over the same time points? I don't usually hear that chemical inhibitors degrade their targets, so that's why I asked all my questions. What is the mechanism of action how your inhibitor should work? (How does it inhibit PKC activity?) If you are concerned that your protein is degraded in response to inhibitor treatment, you could try and use MG 132, a proteosomal inhibitor.

Dear Alessandro,

I don't have a definite answer but here are some (hopefully) educated guesses:

- the inhibitor might have a general/unspecific effect on protein synthesis and/or turn-over - are the PKCs specifically reduced or does this affect other proteins as well? It might not be a trivial task to find suitable reference proteins, as one should consider different protein half lifes when selecting the reference.

- the inhibitor may induce strong changes in the structure of the PKCs upon binding, making them specifically prone to degradation. Does this compound hit other kinases apart from PKCs and are those degraded also? Are the PKCs ubiquitinylated in presence of the inhibitor?

- what about the levels of the PKC mRNAs - do they increase or decrease? In case the levels decrease, you might have to consider an effect of the compound on transcription

Dear Daniel,

thank you for your advices.

Regarding the mRNA expression of the PKCs, I have already controlled and it does not change during the treatment with the inhibitor. However, I was guessing some off-target effects of this compound but I did not think about the possibility that its binding with PKCs can make them easily degradable. It is a very interesting guess, thank you very much. I will try to find it out (maybe using Mg132, proteasome inhibitor).

Dear Alessandro,

I would not be surprised if the apparent degradation of the PKCs is reduced when the proteasome pathway is inhibited. They have to go somewhere, after all.

But it is a quite interesting finding nontheless: One would expect that at least the mRNA of a blocked enzyme increases, as this would be the normal way of a cell to respond to insufficient activity. But it is well possible that this reaction takes longer than your current experimental setup can detect. So it might be worthwhile to extend the incubation times and see what effects on mRNA and protein level can be observed. On the other hand, the longer the PKC-inhibitor is present the more pronounced potential off-target effects might become as well as general toxicity of the inhibitor.