Protein A reduces the protein B levels. At mRNA level there is no change. I thought of checking at translational level. With Protein A and CHX, Protein B level increases. I am confused of giving an explanation. One way could be that Protein A prevents Protein B translation. But when CHX is added, it should be additive, reducing the protein B more, but instead, it is increasing. I checked the mRNA levels of protein too, they are continously increasing in time points.....

No, CHX basically blocks elongation steps in translation, hence used as an inhibitor of protein synthesis. you can treat the cells to block the protein synthesis and follow the kinetics of protein stability. It does not increases protein level

Yes. Cycloheximide, an antibiotic produced by the bacterium, Streptomyces griseus, was shown to be an inhibitor of protein biosynthesis in yeast cells; its activity was later confirmed in mammalian cells. Cycloheximide binds to ribosomes.

Protein A reduces the protein B levels. At mRNA level there is no change. I thought of checking at translational level. With Protein A and CHX, Protein B level increases. I am confused of giving an explanation. One way could be that Protein A prevents Protein B translation. But when CHX is added, it should be additive, reducing the protein B more, but instead, it is increasing. I checked the mRNA levels of protein too, they are continously increasing in time points.....

Thanks a lot..I will carefully go through the paper

We need a little bit of information!

I guess your system consists of cells. A and B are identified proteins, which you are able to measure by some means. Are you measuring protein synthesis in your system from amino acids?

What happens to protein A? Were high concentrations of CHX used? And, the time period for incubation?

This is the explanation for the observed reducing effect of protein A upon protein B.

Have you checked to see what happens when you remove CHX after incubation of protein A with CHX?

CHX exhibits biphasic effects. Have you studied the effects of low and high concentrations of CHX on your system? We had to resort to fairly high quantities of CHX during our studies of in vivo synthesis of mitochondrial outer membrane enzymes.

Certain cells are insensitive to CHX. Perhaps gene transcription and mRNA accumulation in you system is via a hormone, insensitive to CHX. Have you examined the effect of puromycin, which acts by a different mechanism, on your proteins (A and B)? Your end point is the extent of protein synthesis anyway.

What would be the potential effect of sulfhydryl compounds on your system (protein A and CHX)? The action of CHX on peptide chain elongation involves inactivation of a specific sulfhydryl dependent transferase enzyme.

CHX shows two inhibitory effects, apparently based on different mechanisms: 1) Initiation of new peptide chains 2) – elongation of nascent pepides on ribosomes (inhibition of ribosome [polysome aggregation - 60 S subunit)] owing to addition of amino acids).

CHX functions by means of apoptotic cell death.

By definition, cycloheximide should inhibit translation, so if as you say you find an increase in protein B, it cannot be due to an increase in translation. However, the concentration used needs to be considered as well. It is possible though that protein B is regulated at the level of degradation. Have you tried using MG132 to check if A affects the level of protein B even if proteasomal activity is inhibited?

It is quite obvious from your observation that protein A is inhibiting the degradation of protein B. This is the basis of your observation. Suppose if your protein A is a kinase that phosphorylates protein B as a result of which its ubiquitination is hampered and hence the observation. Protein B could also be an deubiquitinase providing this observation. Alternatively, protein A could lead to the degradation of another protein responsible for degrading protein B.

A better explanation is possible if you say what are the types of your two protein (i.e., kinase, dephosphatase, E3 ligase, etc.). If there is no information in this regard, you need to perform certain assays to confirm their types.