Fig. 5.
Ligand-mediated activation of EPHA2 inhibits cell proliferation. (A) M3L2 cells were treated with EFNA1-Fc (1μg/mL) for 24 or 48 h, and cell survival was assayed. (B) M3L2-dCas9-VP64-GFP control cells or EfnA1 guide RNA 1 or guide RNA2 transduced cells were cocultured with EphA2 KO cells or WT cells (C), and the cell numbers were assessed by flow cytometry for GFP+ or GFP− populations. The data are expressed as the ratio of cell numbers from da y0 to day 2 or day 3. (D) M3L2 cells were treated with EFNA1-Fc at 1 μg/mL for time points as indicated, and lysates were immunoblotted with pEPHA2, EPHA2, pERK1/2, ERK1/2, pMEK, and pC-RAF. (E) EFNA1-Fc (1 μg/mL) treated M3L2 cell lysates were immunoprecipitated with GST-Ras binding domain of c-RAF and immunoblotted for KRAS and EPHA2. The PVDF membranes were ponceau stained as a control for loading. (F) EphA2 KO and WT cell lysates were immunoprecipitated with GST-RAF1RBD and immunoblotted for KRAS and EPHA2. Ponceau-stained membrane is shown for loading control. (G) Phospho RAF and PhosphoMEK1/2 in EphA2 KO or WT cells. (H) Mechanistic model of EphA2-mediated tumor suppression in the context of KRasG12D activation.