Figure 3

Crabp1 directly interacts with the RBD of Raf and competes with Ras for interaction with the RBD. (A) Direct protein interaction assay. TnT generated HA-BRaf or HA-cRaf, together with the purified His-Crabp1 was precipitated with anti-HA antibody, and its binding partner His-Crabp1 was detected by anti-His antibody. NS refers to a non-specific band. (B) Mapping the Crabp1-interacting domain of Raf. The constructs of regulatory domain (His-cRaf-(R)) and RBD (GST-Raf-RBD) (Cytoskeleton Inc) used for protein interaction assay are depicted (top). CRD: cysteine rich domain. Ni/NTA beads capturing His-cRaf-(R) or GST-Raf-RBD beads were used to pull down cell lysates containing Flag-HA-Crabp1. Anti-Flag antibody detects increased binding of Flag-Crabp1 to the RBD by RA (bottom). (C) Crabp1 competes with active Ras for Raf interaction. TnT-generated Flag-HRas (WT or G12V) and cell lysates containing Flag-HA-Crabp1 were pulled down by His-cRaf-(R) with or without atRA. Crabp1 was detected by anti-HA antibody, and Ras by anti-Flag antibody from the cut upper region. EV: empty vector. (D) Crabp1 dose-dependent effect in competing with the activated Ras. Increasing amount of Flag-HA-Crabp1 was mixed with the activated HRas (G12V) and pulled down with GST-Raf-RBD (Millipore Sigma) in the presence of atRA. Anti-Flag antibody detected HRas and Crabp1 on the same membrane. (E) atRA dose-dependent effect on Crabp1 competition with the active Ras. Active HRas (G12V) was incubated with increasing concentrations of atRA in the absence or presence of Flag-HA-Crabp1 and pulled down with GST-Raf-RBD beads (Cytoskeleton Inc). The pull-down products were detected by western blots with the indicated antibodies from the different membranes. (F) Direct assay of Raf activation (phosphorylation at S338) in cells provided with or without Crabp1. All blots are representatives of three independent experiments and relative fold intensity is numerically marked.