Figure 3.

Time course of treatment of human MPM cell lines with Sorafenib (64 µM) results in an early decrease in phosphorylation in AKT followed by a marked decrease in ERK 1/2 phosphorylation in whole cell lysates. In vitro cell cultures of H28 (A) and H2052 (B) were treated with sorafenib at a concentration of 64 µM at 37°C, 5% CO₂ for 0 (no sorafenib), 15 min (15'), 30 min (30'), 1 h (1°) and 3 h (3°). Cells were then washed with dPBS and lysed in 1X LDS sample buffer with 2-ME, boiled and run on a 4–12% pre-cast Nupage gel followed by immunoblotting with the following primary antibodies: PDGFR pathway activation antibody cocktail (Cell Signaling) with antibodies to phospho-PDGFR, phosphor-SHP 2, phospho-AKT, phosphor-ERK 1/2 and S6 loading control. These data reveal an early marked decrease in the phosphorylation levels of AKT in response to treatment with sorafenib with near recovery to baseline levels by 3 h of sorafenib exposure. This is followed temporally by a sharp, sustained dephosphorylation of ERK1/2 that begins to recover by 3 h of sorafenib therapy.