Figure 7.

Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.