Fig 2: Generation and characterization of RGD10 peptide-fused RT11-i iMab.(a) Generation of integrin avß3/avß5-targeting RT11-i by genetic fusion of RGD10 peptide, using a (G4S)2 linker, to the N-terminus of the LC of RT11. (b,c) RT11-i and TMab4-i bind to cell surface-expressed integrin a?ß3 and a?ß5. In b, flow cytometric analysis of the cell surface expression levels of integrin a?ß3 and a?ß5 on WT K562, integrin a?ß3-transformed K562, and human tumour cells, analysed by PE-conjugated anti-human integrin a?ß3 and a?ß5 antibodies. In c, flow cytometric analysis of cell surface binding levels of the indicated antibodies, co-incubated at 100 nM with 300 IU ml-1 heparin for 1 h at 4 °C with the indicated cells before analysis. (d) Cellular internalization and co-localization of RT11-i, but not TMab4-i, with the inner plasma membrane-anchored active Ras·GTP in KRasG12V-harbouring SW480 cells. The RasWT-harbouring HT29 cells were also analysed as a control. The areas in the white boxes are shown at increased magnification for better visualization. The arrow indicates the co-localization of RT11-i with activated Ras. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 5 µm. (e) IP of endogenous KRasG12V with RT11 or RT11-i, but not TMab4 and TMab4-i, from endosome-depleted cell lysates of SW480 cells. Images are representative of two independent experiments. In d,e, the cells were treated with 1 µM of antibodies for 12 h before analysis. (f) Inhibition of tumour cell soft agar colony formation by RT11-i compared to that with TMab4-i. Following treatment of cells with PBS, TMab4-i (2 µM), or RT11-i (2 µM) every 72 h for 2–3 weeks, the number of colonies (diameter>200 µm) was counted by BCIP/NBT staining, as shown in the pictures of representative soft agar plates (Supplementary Fig. 7f). The results are presented as percentages compared to the PBS-treated control. Error bars represent the mean±s.d. (n=3). **P<0.01, ***P<0.001; NS, not significant.

Fig 3: Association of Ras and CD68 + cells in angiogenesis. (A) Expression of CD34 in breast cancer cells. (B) Association of Ras expression with MVD number. (C) Association of CD68+ cell number with MVD numbers.

Fig 4: Profiling of the pan-RAF inhibitor LY-3009120 and the paradox breaker PLX-0012. a Structure of LY-3009120, PLX4720, and the Paradox Breaker PLX-0012. Dose-response analysis of LY-3009120, PLX4720 and PLX-0012 with BRET biosensors measuring BRAF kinase domain dimerization b, KRAS-BRAF association d, and BRAF intramolecular interaction f. LY-3009120, PLX4720, and PLX-0012 have distinct propensities to promote BRAF–CRAF dimerization c, to stimulate KRAS-RAF association e, and to perturb BRAF autoinhibition g as measured by co-IP experiments. Cells were treated with 10 µM of each indicated compound. PLX4720 h and PLX-0012 i induce CRAF−BRAF dimerization in a RAS-independent manner. Full-length BRAF−CRAF dimerization was measured in the presence of dominant-negative KRASS17N (gray) or RBD-mutated BRAFR188L and CRAFR89L (RL; blue). Expression of active KRASG12V potentiated the effect of PLX4720 and PLX-0012 on BRAF−CRAF dimerization (orange and cyan, respectively). Each EC50 was the average of three independent replicates. j Maximum pERK signal reported in Table 1 was plotted against its corresponding concentration in each cell line. Each compound is represented by a distinct color: LY-3009120 (red), PLX4720 (orange), and PLX-0012 (blue). To facilitate comparison between conditions, the range between minimal and maximal BRET signals was normalized to 100% in panels h and i. Error bars in dose-response curves correspond to mean values ± s.d. of technical duplicates of a representative biological triplicate. EC50s are the average of at least three independent repeats (Supplementary Data 1). Error bars in j correspond to mean values ± s.d. of biological triplicates

Fig 5: RAF ON-state inhibitors disrupt BRAF intramolecular interaction in a RAS-independent manner. a BRET titration curves demonstrating the specificity of BRAF intramolecular biosensors (BRAFNTR−BRAFKD). Addition of mCherry-KRASG12V robustly impaired the BRAF NTR-KD association, whereas a probe containing the BRAFNTR_R188L mutant was insensitive to activated RAS. b Dose-response analysis of the BRAFNTR−BRAFKD BRET probes with the RAF inhibitor GDC-0879 and a series of MEK inhibitors. c GDC-0879 disrupted the BRAFNTR−BRAFKD complex in co-IP experiments. Anti-BRAF was used to detect the expression of Pyo-tagged BRAFNTR. BRET d and co-IP e show that GDC-0879 equally disrupts the interaction of the BRAF kinase domain with either WT or R188L (RL)-mutated NTR constructs. f Correlation between inhibitor-induced BRAF kinase domain dimerization and the disruption of BRAF NTR-KD interaction. EC50s for each inhibitor in the BRAF-BRAF kinase domain dimerization assay were plotted against EC50s obtained with the BRAFNTR-BRAFKD BRET assay. g The dimerization-impaired mutant BRAFR509H impedes BRAFNTR−BRAFKD disruption induced by type I inhibitors, but does not significantly impede disruption induced by type II inhibitors. GDC-0879 was used at 1 µM in all co-IP experiments. EC50s are the average of at least three independent replicates (Supplementary Figs. 3a, 4g; Supplementary Data 1). To facilitate comparison between conditions, the range between minimal and maximal BRET signals was normalized to 100% in g. Error bars in dose-response curves correspond to mean values ± s.d. of technical duplicates of a representative biological triplicate. EC50s are the average of at least three independent repeats (Supplementary Data 1)