Before getting any conclusion, I thing you should check if you really do not have NFkB in your model by analyzing the presence of the mature active protein, for example by western blotting of cells extracts, and checking both for total protein expression and active form (i.e., phosphorilated p65 NFkB).

I've check the NF-kB expression using RT-PCR. There is no amplication at all for NF-kB gene (40 cycles). However, other genes such as iNOS, IL-6, and TNF-alpha are being expressed. Hence, I'm actually very confused here.

For your information, I've also done an ELISA test using IMGENEX p65 ELISA kit. There was no signal detected at all for p65. That is why I did RT-PCR to check whether there is presence of NF-kB.

From the responses here, you can gather that this is strange.

Have you tried western blotting with a different antibody from that in the elisa?

I think you'd have a hard time publishing a claim of a complete lack of NFKb with the types of evidence you indicate you have.

Yes, I agree with you, Colin. I find it very strange too. But it is possible that the NF-kB expression is depends on the incubation time? For RT-PCR, I've incubated the macrophage cells with 1 microgram/mL of LPS for 24 hours. However, there is no expression detected for NF-kB gene. And for the NF-kB ELISA p65 kits, I've did a range of incubation time, from 30 minutes to 2 hours with 30 minutes interval time. For this, I have also failed to detect the p65 signal.

hey,

for me most interesting is TNFalpha, because the other genes could be just induced because of another stress but so far I know TNFalpha is really just a NFkB target!

I would also check the protein levels with ELISA.

You could also check the IkBa degradation.

Are these BMDMs?

Can you be sure that they do not have working NFkB?

What kind of knockout is it?

Could you also test in addition a NFkB inhibitor??

next step could be to look at p65 levels at the WesternBlot and also the activation of NFkB (phosphorylation)

one more question:

what stimulus did you use?

all the best,

Sebastian

Hi Sebastian! Thanks for your response.

These cells are not BMDMs. I bought these cells from ATCC. It's murine macrophage, RAW264.7 cells (TIB-71). Based on theory wise, I am not very sure whether the NF-kB is really being knock-out from this cell. But based on the results I've got (after experimenting using NF-kB/p65 ELISA IMGENEX kit and RT-PCR), there is no signal detected for p65 and no amplification at all for RT-PCR, although I've run till 40 cycles.

I am not trying to knockout anything from here. Basically, all I want to do is to test the anti-inflammatory effect of a type of drug. But, there is no signal and expression detected for NF-kB. So, I can't proceed with the treatment of the cell with my samples.

I have also thought of purchasing an NF-kB inhibitor from Milipore to block the nuclear translocation of NF-kB p65 and its transcription activity. Then, check the nitric oxide production in supernatant using GRIESS reagent. If nitric oxide is being detected, this shows that it is going through NF-kB pathway. Please correct me if I'm wrong.

A lot of researchers suggested me to check on the p65 level using western blot. But I have already done RT-PCR to check on the gene expression level. In my opinion, I can't really understand why I need to do western blotting since I couldn't detect NF-kB expression in this cell.

For the last question, I am using LPS O55:B5 from Sigma, 1 microgram/ml to challenge RAW264.7 cells for 24 hours.

Regards,

Pui Mun

I also still think that you should also test it with western.

Can you be sure that the primers you are using are fine?

if you use an inhibitor against NFkB you should not get NO or TNFalpha production.

I think best would be to focus on TNFalpha levels (because that is one of the direct targets of NFkB activation (for you NO might me the wrong readout)).

test also the IkBa levels (in western and also in qPCR)

in western you should loose the signal in the first hours and in the qPCR you should get mRNA induction in the first hours.

In addition you should also test the activated NFkB.

all the best,

Sebastian

Yes, I am very sure the primers are working fine as I have tested on another strain of cells, the NF-kB gene was expressed.

Thank you very much for your suggestions. :)

hm...

it wont solve the problem about TNFalpha levels but can you be sure that you got the right cell line? It happens from time to time.

But the thing what you see about TNFalpha and NFkB is still very interesting!!!!

yea....I am also doubt about the cell line that I've choose. The reason I choose this cell line is because I don't need to use interferon-gamma + LPS to activate the cells. I just need to use LPS alone to activate the cell (according to ATCC).

I think you should order new cells. You can take again the same cell line. I just think that they maybe mixed something up and that this is the reason.

but still you got interesting results.

you could also take wt RAW cells and treat them with both stimuli. (in the end this might be more expensive)

Hi Aluru.

I plate 5 million cells in 25cm3 flask and incubate it overnight for attachment. Then, challenge with 1 microgram/ml of LPS and incubate for 24 hours.

For the complete media, it contained of DMEM, fungizone, L-glutamine, penicillin/streptomycin and fetal bovine serum. Do you think that the complete media might contain Vit D3?

And you're right about the LPS-stimulated inflammatory pathway. That is why it is my biggest concern. I thought there might be other pathways (LPS as stimulus) that may induced the production for TNF-alpha and IL-6, but it seems like no other pathways that can produce these proteins without activating the NF-kB pathway.

Anyway, thanks for your response. :)

IL6 is activated by the JAK stat pathway and TNF is activated by MAPK via ErK. So it does not have to be through NFkB.

any body have idea about nfkb2 and il6 link in ductal tumor cells

Hi Pui-Mun Chan

It's been 5 years now. What were your conclusion on the question you asked? one of my lab-mates came across the same.

~ Zeeshan A. Khan

Neurobiology and Molecular Chronobiology Laboratory, University of Hyderabad, Hyderabad 500046, Telangana, India.

Hi guys, I found out you are knowledgeable in cytokine field . I have a question, can IL-6 regulate the production of TNF-a in a cell culture model?

stimulated IL-6 cell line does not show any upregulation of TNF-a and IL-8.Is it what I should expect?