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. 2017 Oct;187(10):2273–2287. doi: 10.1016/j.ajpath.2017.06.009

Figure 4.

Figure 4

Olaratumab inhibits PDGFRα activation and downstream proliferative signaling mediators in HHSteCs in the presence of exogenous PDGF stimulation. A and B: HHSteCs were pretreated with either 300 nmol/L olaratumab or 300 nmol/L human IgG for 30 minutes (m) before 10 ng/mL PDGF-BB exposure for the indicated time periods. Representative Western blots using pooled samples from each time point from three technical replicates show decrease in phosphorylation of tyrosine residues in PDGFRα in cells pretreated with olaratumab, including Y754, Y762, Y849, and Y1018, but not Y742 and Y572/574. Arrowheads indicate the correct molecular weight band. C and D: Representative Western blots using pooled samples from each time point from three technical replicates also show decreased phosphorylation of p38 MAPK (T180/Y182), FAK (Y397, Y576/Y577, Y925), Akt (Ser473), and mTOR (Ser2448, Ser2481) after PDGFRα blockade with 300 nmol/L olaratumab before 10 ng/mL PDGF-BB treatment. E: Decreased phosphorylation of Erk1/2 (T202/Y204) and downstream transcription factor Elk1 (Ser383) was also observed in similar representative Western blots. 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, hour; NT, nontreated.