LPS is a very strong stimulus .... I agree with AP-1 and NF-kB. If you co-stimulate with interferon-g you should analyze in addition STAT1/3, IRF-1 and NFAT. You could analyze the promoter of inflammatory target genes by using the software of genomatix [http://www.genomatix.de/] ... than you know if one of the above mentioned TFs is responsible for the activation of pro-inflammatory gene expression.

Would u go on study on PRR?

Hello,

you can investigate the Jak/Stat pathway and in particular STATs transcription factors.

Hi,

LPS would obvious activate the MyD88/IRAK1 dependent pathway, so NF-kB is your obvious bet. This pathway also stimulates MAPKs such as ERK, JNK1/2, p38 and MEK1/2. These often activate AP-1.

IFN-g stimulates a different pathway - the JAK/STAT pathway. In particular I would look at STAT1, via JAK1/JAK2.

Hi, look for our favorite CEBPD which we and others have shown that it is an initiator as well as an amplifier of LPS signaling. See Balamurugan et al,, Nature Communications 2013, Litvak et al Nature Immunology, 2009. We show that CEBPD acts upstream of TLR4, NF-kB and STAT3

Tbet for Th1 is ideal one

additionally in microgilai cells it has been shown very recently that CEBPD is induced at the early time points in response to inflammatory stimuli LPS,

Valente et al, 2013

Thanks for all the replies! I will study both LPS and IFN-γ signaling and decide which transcription factors I will use.

Hi there, it depends a little bit on the cell type and the differentiation status of the cell. NF-kB, NFAT, AP-1, and Stat3/5 are all also TFs that get activated early on in T cells. In microglial cells, maybe also IRF8 might be interesting. In addition, maybe also Runx and Nrfs or Klfs. Good luck with the experiment!

Few well established transcription factors are NF-kB and AP-1 as also mentioned by Martina..

Many transcription factors are activated by LPS at early stages in the inflammatory response, but at least TLR4, NF-kB, STAT-3/5 and AP-1, should be investigated. The activation usually requires phosphorilation pathways as upstream. Then, activation of kinases and phosphatases should be investigated as well,

HI, like everyone else I would try AP-1 as my first target apart from NFkB which you are already considering. Also, JAK/STAT pathway specifically STAT3 (good antibodies from Cell Signalling). Since you are stimulating with IFNg and interested in inflamation I would look into IRFs (interferon response factors) there are a few but I would look specifically for your system. These options should give you a better view as these pathways activate each other in some way or another. Good Luck.

Hi Martin,

All answer are very good. However, an important point is to know on the particlar cell that you work. There is factors higly expressed in some cells, but no in others

I would use a PCR array that relates to your study area. Qiagen has many to choose from.

T-bet and JAK/STAT

You are right NfkB is your first bet and second AP-1. However, since different pathways are involved in different cell type (and of course if the money is not an issue) I would go global analysis

For a global analysis before focusing on the most altered thanscription factor

c-Jun, AP-1 are the best candidates....specially c-Jun phosphorylation (good antibodies are available) is 1 of the earliest events documented.....

Dont forget kinases. They may not be the "classical" transcription factors, but there is enough evidence now suggesting that they actually may act faster (and potentially more direct) compared to transcription factors.

Depends on animal or cell line you work and on model of inflamatory reaction you use

LPS is a very strong stimulus .... I agree with AP-1 and NF-kB. If you co-stimulate with interferon-g you should analyze in addition STAT1/3, IRF-1 and NFAT. You could analyze the promoter of inflammatory target genes by using the software of genomatix [http://www.genomatix.de/] ... than you know if one of the above mentioned TFs is responsible for the activation of pro-inflammatory gene expression.

LPS signals mainly through NFkB (check the literature about TLR4), and IFNg - in addition to JAK/Stats worth looking at IRF3, 5 and 7.

By using DNA arrays or similar techniques which measure differences in RNA levels you will miss the rapid activation of transcription factors which are pre-formed (like NF-kB and NFATs). Do not forget that most early response gene are "open" and show a low increase in expression level, contrary to chemokine or other genes which are more than 100 fold increased by LPS. If you have enough material, study the nuclear translocation of NF-kB and NFATs (e.g. in EMSAs, Western blots). Moreover, phoshorylation studies - as mentioned by others - should also be done.

Stat family of TF, SP-1 and c-myc

NF-kB, AP1 (c-jun/ATF2) and IRF3 should be activated by phosphorylation after LPS stimuli (The two first are MyD88-dependent, while IRF3 is TRIF-dependent). There are good antibodies to both unphosphorylated and phosphorylated/activated forms of all these TFs. In addition the kinases responsible for phosphorylating these TFs are themselves activated by phosphorylation and are good candidates for WBs themselves. There is considerable cross-talk between these pathways and different cells utilize different variants of these pathways, so remember that you might be looking at something that is not very well defined in the literature.

i think you could stimulation by LPS or IFN-r(i think LPS better ,you can get more),then do RNA seq,you will find a lot of gene,then analysis the pathway involved.good luck.

LPS Highly upregulates NF-KB. The next candidate should be AP-1

Does anybody have a decent recent review on TLR4/IFN signaling to recommend?

Check out "Toll-like receptors and innate immunity" and "Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity", both by Akira. Good starting points and they should point you to more specialized papers about TLR4.

phospo-NFKB and p-STAT1 might be detected 15-30 min after stimulation, both following stimulation with LPS and p-STAT1 after IFNg. Measuring them by FACS might be relatively fast and easy.

See papers published by a former student of mine - Sergiu Catrina and his wife

I think you have most of the answers from previous posters. NFkB is activated within 5-10 min of LPS stimulation in my own hands and we've published as well. Both AP-1 and NFAT will also be activated and STATs will be activated in response to IFNg. However, what is your readout? You can look at TF activation by looking for their phosphorylated forms in WB. But if your readout is gene expression, which typically starts by 20-30 min and then peaks by 1.5-2 hrs, then it gets a bit complicated. If say, you're looking at IL6 expression, most of the TFs are gonna upregulate it (Look at the Chip seq data of IL6 promoter, most of the known TFs bind). So your inference regarding a specific pathway/TF will depend on the way you ask the question.

Dear

Theodore Kapellos

Your topic is good. Apart from NFkB , look for HIF1 alpha , and Nrf2 transcriptional activity under inflammatory conditions. You also determine some of the Oxidative stress parameters under inflammatory condition, which all together will give you a complete picture of infalmmation in microglial cells under LPS treatment.

All the best.

With warm regards

from Dr. Sarada SKS

Scientist "E"

DIPAS, DELHI-54

INDIA.