Tour-de-Force

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