Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Kinase assay AlphaScreen® PhosphoSensor KitsUsing these kits one is able to test the activity of various kinases in phosphorylation a particular substrate.This method does not require us to identify particular phosphorylation sites using antibodies however,antibodies will be required in order to purify and isolate the various substrates we are interested in. TheAlphaScreen® PhosphoSensor Kits requires the substrate (in this case NRF-1 or 2) to be biotinylated. Asthe substrate is phosphorylated (by the specific kinase in the assay) a streptavidin donor andPhosphoSensor acceptor bead capture the substrate, causing the PhosphoSensor acceptor bead to emitlight. This emission can be observed using most imaging systems providing a quantification of thephosphorylation activity of the specific kinase with respect to the substrate being examined.Mutations: The role of localization of Cyclin D 1 /cdk 4 in NRF regulationWe will mutate the following site: T286A (Wang et al., 2006). In this study it was found that the continuedlocalization of Cyclin D1 in the nucleus resulted in continued inhibition of NRF-1. However, whether thisdown-regulation is still observed when PRC is activated during the cell cycle has not been established.RNA isolationCells will be treated with TRIZOL Reagent (Invitrogen) to isolate RNA according to manufacturer’sinstructions.RNAiCustom siRNAs designed for PGC-1α, PGC-1β, PRC, and MAP2k6 knockdown will be ordered throughApplied Biosystems with the target mRNA sequences obtained from RefSeq (PGC-1α: NM_013261 ;PGC-1β: NM_133263; PRC: NM_015062). Positive and negative controls for siRNA are: Silencer®GAPDH siRNA and Silencer® Negative Control #1 siRNA (Applied Biosystems). Vectors containing thesiRNA of choice according to the experiment will be inserted into the cell using the pSUPER vector(Oligoengine 2.0) designed by Thijn R. Brummelkamp et al. A Western blot will then be performed tocheck whether the vectors have been successfully expressed. Applied Biosystems TaqMan® GeneExpression Assays specific to PGC-1α, PGC-1β and PRC will be used to detect the RNAi effect.MAPK-p38 inhibitionTo inhibit the MAPK-p38 pathway, we will be using SB220025 MAPK INHIBITOR Multiple (BioSource)(Invitrogen). SB 220025 is a potent inhibitor of p38 MAPK for elucidating p38 MAPK's role in signalingpathways.MAPK Erk inhibitionInhibition of the MAPK Erk ½ pathway will be achieved using the MEK Inhibitor U0126 (Promega). U0126Inhibits both active and inactive MEK1,2 unlike PD098059 (the other possible inhibitor for this pathway)which only inhibits activation of inactive MEK (1,2). MEK Inhibitor U0126 results in downstream inhibitionof Erk 1 and Erk 2 mediated responses.RT-PCR SystemFor our research we would like to apply for an extra grant to purchase the Light Cycler 2.0 from RocheDiagnostics. There are two Light Cycler systems; the 2.0 and 480; the difference being in the samplenumber one is able to process at one time. The Light Cycler 480 is a larger system for 96- and 384- wellthermal cycler units. Since our sample size is not that big we opt for the Light Cycler 2.0 which has 32 wellthermal cycler units, which is enough for our experiments.What sets the Light Cycler apart from other systems is that it allows for very rapid running of samples in atime span of 30 minutes. Furthermore, this technology has improved sensitivity through rapid cyclingwhich increases the signal to noise ratio and protects against the accumulation of unwanted PCR products.In comparison to the BioRad iCycler iQ Real Time PCR System supplied by Bio Rad, the Light Cycler hassix fluorescence detection channels that measure fluorescence at 530, 560, 610, 640, 670 and 750 nm.The BioRad iCycler iQ Real Time PCR System only has a three color analysis systems. Moreover, theLight Cycler 2.0 comes with optimized hardware components, which allow for rapid, precise, temperatureregulation for maximum reproducibility as well as providing us with an unlimited choice of detection format.SCI 332 Advanced Molecular Cell Biology Research Proposal 114