Fig. 4

Inducible expression of different RON mutants in T47D cells and analysis of downstream PI3K/mTORC1 signaling. a Schematic presentation of RON kinase structure is shown in the left panel. Mutations that were introduced to RON are shown in the right panel. Mutated residues are highlighted in pink. b Inducible expression of RON mutants in T47D cells at the protein (upper panel) and mRNA level (lower panel). T47D cells transduced with different RON mutants in a Tet-inducible lentiviral plasmid were treated with 500 ng/ml doxycycline for 48 h, and analyzed by Western blot. Equal level of RON expression is apparent among the mutants, which is comparable to RON wild type (WT). Graph shows qRT-PCR analysis of T47D cells expressing RON mutants in the presence and absence of doxycycline (500 ng/ml) for 48 h with primers specific to RON. GAPDH was used as the internal control. c Signaling analysis of the PI3K/mTORC1 pathway in T47D cells expressing RON WT and mutants. T47D-RON WT and mutants were treated with 100 ng/ml doxycycline for 48 h, followed by serum starvation for 24 h. Western blot shows robust activation of AKT, P70S6K, and rpS6, as readouts for activation of PI3K and mTORC1 kinase pathways, in Mutant A compared to the other mutants. GAPDH was used as the internal control. See also Supplementary Figure S8 for signaling analysis of mutants, and Supplementary Figure S9 for the detailed signaling analysis of RON-Mut A in the context of MSP-dependent and -independent RON activation. Line indicates the separation between lanes on the same Western blots. PSI plexin–semaphorin–integrin, IPT immunoglobulin–plexin–transcription, TM transmembrane