What is the meaning of NF-kB (transcription factor) "activity"?

Question

This article discuss about the NF-kB activity and its mathematical modeling Understanding NF-κB signaling via mathematical modeling. The NF-kB is a transcription factor mainly involved in the regulation of inflammatory genes. However, I do not really understand this: what do we mean by NF-kB activity ? For instance, if we consider this graph (that is figure 1A in the paper that I linked):

by NF-kB activity on the vertical axis, do we mean the number of RNA molecules produced by the target gene regulated by the NF-kB over time ? Or it depends on the kind of experiment ? In the article it tells: this prediction was confirmed by using EMSA on wild-type cells. What does EMSA effectively measure ?

P.S. The idea that by activity I mean the number of RNA molecules produced comes from the fact that I read some thesis works about NF-kB activity in which Cignal Lenti Reporter System (QIAGEN) is used for the experiment and I read in Reporter Gene Assays that a reporter gene assays are typically used to measure the regulatory ability of an unknown DNA-sequence. This is done by linking the unknown promoter sequence to an easily detectable reporter gene whose product can be easily detected and quantifiably measured. However I do not really understand how EMSA can experimentally give us information about NF-kB activity (maybe because I did not understand how it works).

I thank you in advance.

Answer 1

The word activity has at least two meanings in biochemistry. One is chemical activity as in the effective concentration of a chemical. This comes up a lot because biomolecules often behave non-ideally.

The other sense, relevant here, originates in the description of enzymes. As catalysts, enzymes accelerate the rates of chemical processes. Enzyme activity characterizes the rate of an enzyme catalyzed reaction, so these activities are quantities that include time dimensions (Cornish-Bowden, p. 81).

This leads to a definition of the activity of a transcription factor, a biomolecule that catalyzes the initiation of transcription. The activity of a transcription factor is its enhancement of transcription (which, since uncatalyzed transcription has an effective rate of 0, is the same thing as the observed rate of transcription).

Usually this is only indirectly measured, and usually the absolute activity (in, say, transcription initiations per cell per hour) isn't needed, only the relative activity. That's why activity is often illustrated on axes with normalized/absent units; the raw units are often arbitrary. So, yes: activities in this case could be literally measured by the number of mRNAs transcribed when NF-κB is activated. But anything proportional to that mRNA population is also suitable as a measure of activity.

From the reference in question:

NF-κB enters the nucleus, denoted by the suffix ‘n' (k1). This leads to synthesis of IκB mRNA transcript, denoted by the suffix ‘t' (tr2).

So, synthesis of the IκB transcript is the main response variable they're interested in tracking in term of NF-κB activity. But that's not the definition of activity, just a convenient measure of the promotion of transcription. NF-κB promotes many transcripts, not just IκB!

In 5, electrophoretic mobilility shift assays are referred as follows:

As time-course data, derived from electrophoretic mobility shift assays (EMSAs), indicated that the three IκB proteins had differential dynamic control, we set out to construct a mathematical model of NF-κB signaling to study the specific roles of each IκB isoform in regulating the temporal control of NF-κB (Hoffmann et al, 2002)

A biomolecule has a certain mobility in this experiment and the mobility shifts when the shape or charge distribution of the biomolecule changes. So here the raw signal is the migration distance of a given band (somewhat arbitrary as a measurement, but proportional to activity).

I have not yet checked the Hoffmann et al 2002 paper. But if we assume that, here, "EMSA" summarizes experiments 1) comparing nuclear and whole cell extracts (key for deciding that NF-κB is activated) and 2) using Western blotting for confirming protein bands, perhaps the "confirmed by EMSA" phrases are less opaque.