Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007considering that we are looking at many compounds. It requires careful planning and design to perform asuccessful RT-PCR, and as we are using this method to quantify quite a number of different compounds, itwill probably take multiple weeks or months.Fluorescent-activated Cell Sorting (FACS)FACS will be used to measure the relative changes in mitochondrial mass throughout the cell cycle. Wewill be utilizing the laboratory facilities at the University of Utrecht.As the target(s) of a single probe for mitochondria may not be representatives of the changes inmitochondrial mass, different probes will be used. The fluorescent probes need to be membrane potentialindependent and mitochondrial uptake of the fluorescent probe should also be membrane potentialindependent. Both MitoTracker Green FM and NAO (10N-nonyl acridine orange) have these propertiesand will be used as the fluorescent probes in FACS. The targets of the former probe are a subset ofmitochondrial proteins that have free thiol groups of cyteine residues in mitochondrial proteins (Presley etal., 2003), while the target of the latter probe is cardiolipin, an inner membrane component of mitochondria(Petit et al., 1992)Western BlottingFor the Western Blot analysis to detect protein expression of the different compounds, specific primaryand secondary antibodies have to be ordered (Invitrogen).In general, it is better to use monoclonal antibodies in Western Blotting. Apart from the obvious reasonthat monoclonal antibodies have a higher specificity than polyclonal antibodies, they also ensure a lowerbackground and less non-specific detection.Antibodies against PRC and NRF-2 however are commercially unavailable. However, a possible way totarget NRF-2 is by targeting the α subunit, which is homologous with GABPA (GA-binding protein chain α)and does have antibodies against it. This means these antibodies will also target NRF-2α, which has beenshown in a research studying the function of NRF-2 (Ongwijitwat & Wong-Riley 2005). As the genetic codeof NRF-2 remains unknown or incomplete, at this point we are unable to synthesize antibodies againstNRF-2 directly. An antibody against PRC will be synthesized (Invitrogen).The negative control for both RT-PCR and Western Blot will be β-tubulin, because its expression levelsare hardly affected by changes in the cell, and β-tubulin has been successfully used as a control inprevious research in the same field (Wang et al. 2006). The antibody for β-tubulin will be bought fromInvitrogen Corporation and the primer will be obtained from Superarray Bioscience Corporation.Co-ImmunoprecipitationRegarding binding between Cyclin D1 and cdk4 and 6, the antibodies against these compounds used forthe Western Blot can also be used for co-immunoprecipitation.Phosphoprotein and phosphopeptide analysis: Identification of the nature and extent of phosphorylation ofNRF-1 and 2 by Cyclin D1/cdk4 and PRC: Phosphorylation detection of NRF-1 and PRCPerkinElmer Phos-tools kit will be used along with PerkinElmer Phos-tag stains to highlightphosphorylated proteins due to their high binding affinities with phosphorylated serine, threonine, andtyrosine residues. Additionally, for this method it is required to enrich the phosphopeptides for adequatedetection using PerkinElmer Phos-trap.Using these kits, as described by the manufactures, we will be able to identify if and at which sites NRF-1is phosphorylated by Cyclin D 1 /cdk 4 . Using this method requires one to first identify the protein of interestusing 1D PAGE analysis. Following this, staining with PerkinElmer Phos-tag will reveal if the protein ofinterest (in this case NRF-1) is phosphorylated. This stain does not target specific phosphorylation sitesbut binds with the phosphomonoesters of tyrosine, serine and threonine. Following this PerkinElmer Phostrapis necessary to enrich the phosphopeptides and the protein is then analyzed using massspectrometry to identify the sites of phosphorylation. We will then do the same process to establish thenature of phosphorylation of NRF by PRC.SCI 332 Advanced Molecular Cell Biology Research Proposal 113