The residues serine 479 and serine 499 of human Raf-1 are thought to be important for its activation. Three different constructs were made to investigate the function of these residues. All were chimeric constructs with Raf-1 tagged with GST at the N-terminus. The constructs included a wild-type Raf-1, Raf-1 with serine 497 and 499 replaced with alanine and Raf-1 with serine 497 and 499 replaced with aspartic acid. These constructs were ectopically expressed in HEK 293T cells which were treated plus and minus EGF. A GST-pulldown was performed, and Raf-1 activity was determined in vitro.

a. Why was it necessary to perform an immunoblot for GST in this experiment?

b. Which construct exhibits constitutive activity? Explain how you can tell from the data above.

c. Speculate on why this construct is constitutively active.

d. Propose a way that Raf-1 activity was measured in this experiment.