Fig. 3. Activation studies of the autoinhibited BRAF/MEK1/14-3-3 complex.
a–c The time course of phosphorylation of ERK2 by the purified BRAF/MEK1/14-3-3 complex, with or without the addition of the indicated KRAS construct and/or liposomes, is measured by western blotting with an anti-pERK1/2 antibody. Integrated band intensities, normalized to the intensity of the autoinhibited complex alone at the 80 min time point, are shown under the blots. a Activity of the autoinhibited complex alone, or with the addition of either GMP-PNP loaded KRASGTPase or KRAS4b-FME at a 1:1 molar ratio. b Activity of the autoinhibited complex alone or with the addition of phosphatidylserine (PS) liposomes, KRAS4b-FME, or both liposomes and KRAS4b-FME, as indicated. Liposomes were added to a final concentration of 0.1 mg/ml, and GMP-PNP loaded KRAS4b-FME was added in a 2:1 molar ratio to the autoinhibited BRAF/MEK1/14-3-3 complex (5 nM). c Comparison of the relative activity of the BRAF/MEK1/14-3-3 complex in the autoinhibited vs. active, dimeric state, with or without the addition of KRAS4b-FME. The samples for the autoinhibited complex with and without KRAS4b-FME are aliquots of the same reaction as the corresponding samples in (b), re-run on the same gel and blot with the active dimer reactions to allow comparison of their relative activities. Note that the exposure of the blot in (c) (5 s) is much shorter than that in (b) (2 min). Original uncropped blots are provided in the Source Data File. Experiments in (a–c) were performed three times each with similar results.