FIGURE 3 from Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor–positive Breast Cancers for Targeted Treatment

NF1 binds palbociclib-conjugated bead directly. A, Reference cell lysate known to contain up to 93% of kinases in human genome (see Materials and Methods; ref. 16) was incubated with beads conjugated to indicated single kinase inhibitor. A heat map was generated to show protein levels (iBAQ) on the bead as measured by MS. B, Purified NF1 was incubated with abemaciclib or palbociclib conjugated beads and the bound NF1 was analyzed by MS (left) or by gel electrophoresis after Flamingo staining (right). SecPH domain from NF1 was examined as the negative control. C, Full-length and truncated NF1 proteins were pulled down by drug-conjugated beads and analyzed by gel electrophoresis as in B. D, Purified NF1 (500 ng) was incubated with palbociclib beads and digested by trypsin. The products were serially diluted (1:10) before SureQuant.