(1) It has been proposed that transcription factor NF-kB can bind to the promote
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Question
(1) It has been proposed that transcription factor NF-kB can bind to the promoter of IL-1or TNF- gene. Please provide your interpretation on the results from western blot analysis of IL-1, TNF-, and NF-kB in macrophages. (5 pt)
(2) The expression levels of NF-kB total protein (NF-kB P65) was not changed with LPS treatment, while the expression of phosphorylated NF-kB (NF-kB p-P65) was increased with LPS treatment. Please provide your explanation for this, in particular in the context of posttranslational modification of protein and its functional significance. (5 pt)
(3) Why is it necessary to compare proteins of your interest with -actin? Please explain why the level of b-actin protein was not changed with LPS treatment. (5 pt
Chegg Study l Guided Solu x HW2.pdf 3 14 5. You are doing a lab research to examine how the recognition of bacteria by macrophage contributes to the host defense. It has been shown that the secretion of pro-inflammatory cytokines IL-1 and TNF- is critical for eliciting innate immune response for host defense and you want to examine whether a transcriptional activator, NF-kB, plays a critical role in the response. For this, you have cultured macrophages and treated the cells with bacterial endotoxin LPS (Lipopolysaccharides) to mimic bacterial infection to macrophages. After 8 hours of treatment, proteins were extracted from the cells and the expression of proteins were analyzed by westen blotting. The results were compared with macrophages in the absence of LPS treatment ) ILI- -actin NF-x B p-P65 NF-KB P65 -actin LPS-+ LPS- + LPS -+ Based on your western blot results above, please provide your answers to the following questions. (1) It has been proposed that transcription factor NF-kB can bind to the promoter of IL-1 or TNF-a gene. Please provide your interpretation on the results from western blot analysis of IL-1, TNF-, and NF-kB in macrophages. (5 pt) 2/25/2018Explanation / Answer
1.
From the first and second gels, we can conclude that IL1-beta and TNF-alpha are induced in the presence of LPS. The third gel shows that the overall protein level of NF-KB did not change with LPS treatment but the fraction of phosphorylated NF-KB is increased in the presence of LPS. This shows that NF-KB is phosphorylated in response to bacterial infection and gets translocated to the nucleus to upregulate its downstream target genes such as IL-1 and TNF.
2.
The presence of LPS triggers a signaling cascade which ultimately results in the activation of NF-KB by phosphorylation. Here, the signaling is aimed at increasing the active form of NF-KB rather than increasing the protein level as such. Phosphorylation is a post-translational event mediated by kinase or phosphorylase.
3.
We always have to compare the test results with a control. In this case, if we want to say that IL1 or TNF levels are increased, we must have control which does not show an increase in level upon treatment with the LPS. Here, actin is used as the control. Actin is a constitutive gene and is not directly involved in immune response. So, it is not triggered by the presence of LPS.
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