Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007per 10 6 cells for 60 minutes on ice. After washing, cells will be incubated with 1 µg of Alexa Fluor 488-conjugated goat anti-rabbit antibody per 10 6 cells for 30 minutes on ice, washed and re-suspended in PBS.Cells will be prepared for flow cytometry following the protocol accompanying the anti-pSer10-histone H3antibodies. FACS will be carried out according to the protocol to count cells containing phosphorylatedhistone H3.Materials:• Nocodazole (Sigma)• Hydrogen peroxide (Sigma)• Ethanol (Sigma)• PBS containing 0.1% Triton X-100 (Invitrogen)• anti-pSer10-histone H3 anti-rabbit antibody (Cell Signaling Technology)• Alexa Fluor® 488 F(ab') 2 fragment of goat anti-rabbit IgG (Invitrogen)FACS for cell cycle phase checkPreparation of cells for FACS will be performed as described by Ezhevsky and colleagues (1997): Cellsarrested in prometaphase will be washed with PBS, fixed in 70% ethanol overnight at -20 °C, andrehydrated with PBS containing 0.1% BSA, RNAase A (1 µg/ml) and propidium iodide (10 µg/ml), 20minutes before analysis with FACS. For FACS procedure, the protocol accompanying the machine atUtrecht University will be followed.Appendix 3.2: Immunoprecipitation Kinase AssayImmunoPrecipitation Kinase Assay:Immunoprecipitation will be applied as explained by Ezhevsky et al. (2001) using 2 µg of antibodiesmentioned below.Antibodies:− Cyclin D1 antibody (ab7953) (Abcam) binds to D1 and D2− Cyclin A antibody (ab53054) (Abcam) binds to A1 and A2− Cyclin D1 antibody (ab7953) (Abcam) binds to D1, weakly binds to D2− 50 µg of protein A beads will be usedKinase assay will be applied to cell extracts obtained from immunoprecipitation as described in the manualof Trulight Universal Kinase/Phosphatase Assay Kit (Calbiochem).Substrates:Histone H1 (Calbiochem) for cell extracts incubated with antibodies against cyclin A and cyclin Bglutathione transferase (GST) C terminus pRb (Calbiochem) for cell extracts incubated with antibodyagainst cyclin D assay.2 µg of substrate will be used together with 50 µg cold ATP (Amersham). Reactions will be performed for30 minutes at 30°C.Measurement of Kinase activity:Kinase activity will be measured as explained in the kit manual. Statistical analysis will show the activity ofthe kinase as a percentage which makes it possible to compare kinase activities.Appendix 3.3: Western BlotWestern blot will be done as explained by Ezhevsky et al. (2001) using antibodies mentioned below as aprobe. Beta Actin antibody (Abcam) will be used as a loading control.a) Antibodies:− Cyclin A antibody (ab53054) (Abcam) binds to A1 and A2− Cyclin B2 antibody(X29.2) (Abcam) binds to B1 and B2b) Phospho-specific antibodies:SCI 332 Advanced Molecular Cell Biology Research Proposal 108
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007−−−−Phospho-specific anti-cdk1 antibody (pThr14, pTyr15) (Calbiochem) binds to cdk1, phosphorylated atThr14 and/or Tyr15Cdk2 (phospho T160) antibody (Abcam) binds to cdk2 phosphorylated at T160Anti-Cdk2 (Abcam)Anti-Cdc2 (Abcam)Appendix 3.4: 35 S pulse-chase experiments35 S pulse-chase experiments:35 S pulse-chase experiments and Western Blot will be performed as described by Toby et al. (2005).Antibodies used:Cyclin A antibody (ab53054) (Abcam) binds to A1 and A2Cyclin B2 antibody(X29.2) (Abcam) binds to B1 and B2Measurement of Incorporation:Incorporation will be observed in SDS-Page and quantified by phosphorimager. Phosphorimagermeasures intensity of photon emission which is an indication of how much the protein is translated.Appendix 3.5: Co-immunoprecipitationCo-immunoprecipitation:Co-immunoprecipitation experiment and Western Blot will be performed as described by Havens et al.(2006).Antibodies used:Anti-Cdc27 AF3.1 antibody (Santa Cruz) conjugated with Protein A-Sepharose beads.Beta Actin antibody (Abcam) will be used as a loading controlAppendix 3.6: Cycloheximide treatmentThis experiment will be performed as explained by Havens et al. (2006)Cells will be harvested at three intervals for western blotting:1. Before treatment with cycloheximide2. 1.5 hours after treatment with cycloheximide3. 3 hours after treatment with cycloheximideAntibodies:− Cyclin A antibody (ab53054) (Abcam) binds to A1 and A2− Cyclin B2 antibody(X29.2) (Abcam) binds to B1 and B2− Beta Actin antibody (Abcam) will be used as a loading control.References:“Bio Currents Research Center Protocol - Electrode Construction” Retrieved on 20 November 2007“Bio Currents Research Center.” MBL, Woods Hole, MA. Retrieved on 20 November 2007Demaurex, N. (2002) “pH homeostasis of cellular organelles”. News in physiological sciences 17:1-5Ezhevsky, S. A., A. Ho, M. Becker-Hapak, P. K. Davis, and S. F. Dowdy (2001) “Differential regulation of retinoblastoma tumorsuppressor protein by G1 cyclin-dependent kinase complexes in vivo.” Mol. Cell. Biol. 21: 4773–4784.Filippin, L. et al. (2005) “Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix” Cell Calcium37:129-36.Foster et al. (2006) “Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress andneurodegeneration”. Progressive neurobiology 79:136-171Havens, C. G., A. Ho, Yoshioka, N., and Dowdy, S. F. (2006) “Regulation of Late G1/S Phase Transition and APC by ReactiveOxygen Species.” Mol. Cell. Biol. 26: 4701–4711.SCI 332 Advanced Molecular Cell Biology Research Proposal 109
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