When they are activated, te G protein exchanges GDP for GTP. A a result of this dissociation of the GTP-bound α and β/γ subunits, anumber of diverse signalling cascades are triggered. Rceptors attached to diverse heterotrimeric G protein subtypes can use different scaffolds to activate the small G protein/ MAPK cascade such as Tyr kinases. GCRs may also utolize PLCβ as a mediator in the activation of PKC and CaMKII, wich can either stimulate or inhibit the MAPK pathway downstream. I the experiment above EPGF was a positive control since the HTl cells normally over express HER2 which is an EGF receptor.
Te negative control consisted of untreated cells which lacked the external stimuli to induce signal transduction thus there was limited proliferation. O the basis of the evidence in fig 1 and using a diagram, ientify the pathway(s) that is (are) most likely to lead to cell proliferation following treatment with the MF8 ligand. Gve clear reasons for your answer. Aditionally, poliferation in the presence of MF8 was observed in H89 which is a Hwever, sgnal transduction will not stop since PKA and EPAC are responsible for activation of RAP-1 thus it continues to proliferation stage.
Poliferation was also observed in PMA due to its ability to induce PKC activation thus continuation of the signal transduction. O the other hand minimal proliferation was observed in the PD98059 which is an ERK inhibitor. Tis is due to the fact that when ERK is inhibited it will not enter the nucleus where transcription occurs. I was also observed that little proliferation was recorded in PKC Tis is because it inhibited RAS GAP which in turn inhibits Rasthus stopping the signalling process.
I addition to the PCR/Western blot techniques flow cytometry can also be used to analyse signal transduction pathways in culture cells by detecting phosphor-epitopes. Te Stat5 phoshorylated on tyrosine can be used as a cell based measure of Bcr/Abl activity in chronic myeloid leukemia cell line signalling. Tis experiment can be repeated with kinase inhibitor such as STI571. Tis kinase inhibitor blocks transcription thus preventing proliferation of. ..
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