Figure 4: SHP2 inhibition promotes tyrosyl phosphorylation of wild-type and oncogenic Ras leading to inactivation of Ras and downstream signalling.

(a) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. (b) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. (c) HEK293 cells transfected with the indicated plasmids were lysed, pulled down using Raf:RBD and immunoblotted with the indicated antibodies. (d) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. (e) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml⁻¹ of EGF, lysed, pulled down using Raf:RBD beads and immunoblotted with indicated antibodies. (f) Equal number of RasB8p3 astrocytes were plated in 96-well plates in sextuplicate and treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h and alamar blue assay was conducted according to the manufacturer’s instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. *P<0.05 Student's t-test compared with DMSO control. (g) Equal number of RasB8 astrocytes were suspended in agar matrix, and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed according to the manufacturer’s instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. *P<0.05 Student's t-test compared with DMSO control. Representative images are also shown on the left panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; PD, pull down; WCE, whole-cell extract.