Your findings indicate that substance X arrests the cells after crossing the restriction point and before reaching S-phase.
We would like to suggest a molecular mechanism for this phenomenon. Therefore, we will now inspect an illustartion depicting the various molecules that participate in each phase of the cell cycle.
 
9. Which of the following mechanisms may explain these results? (Choose TRUE or FALSE) :
A. Elevated activity levels of CDK4-cyclin D.
B. Inhibition of CDK2-cyclin A activity.
C. Partial phosphorylation of pRb.
D. Inhibition of CDK2-cyclin E activity.
Choose the right answer/s and explain. 

10.

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You have decided to perform the following experiment:

  1. Cells are synchronized to G0 by starvation.
  2. CS is than added for 16 hrs.
  3. Cells are exposed to PBS or substance X at t=0-8 or at t=8-16, and all cells are harvested at t=16.
  4. Anti CDK2 and anti CDK4 antibodies are used to immunoprecipitate CDK2 and CDK4 immunocomplexes.
  5. The kinase activities of these complexes are assayed in-vitro using histone H1 (a substrate of CDK2) or  pRb (a substrate of CDK4) respectively. The kinase is precipitated with specific antibodies, incubated with a substrate and radioactive phosphate. After incubating this reaction at the appropriate temperature (37oC) it is run on an Acrylamide gel. The gel is fixed, dried and exposed to a film or phosphorimager plate. If the kinase is active the substrate will be radiolabeled.

    6.  
  6. In addition, you test by western blots the levels of CDK2 and CDK4 in the immunocomplexes to see if you have equal amounts of the enzymes in all samples.
Here are the results:
Legend
Kinase activity
Western blot
11. You may draw the following conclusions (true or false):

A. Substance X does not alter CDK4 levels in the immunocomplex but reduces its kinase activity.
B. Substance X does not affect CDK2 or CDK4 levels in the respective immunocomplexes.
C. Substance X reduces the kinase activity of the CDK2 containing immunocomplex at t=8-16.
D. CDK2 kinase activity in control cells is lower at t=8 than at t=16.