Western Blot Analysis of Akt and PDGFRβ

Sample preparation was performed as described previously.(14 (link)) Briefly, cells were lysed in TENSV buffer (50 mM Tris–HCl (pH 7.5), 2 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1 mM Na₃VO₄, 1% NP-40, 0.1% aprotinin, and 2 mM phenylmethylsulfonyl fluoride), and electrophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were transferred to a membrane and immunoblotted with an anti-Akt (pan) monoclonal antibody (mAb) (clone C67E7, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Ser473) mAb (clone D9E, Cell Signaling Technology), anti-PDGFRβ polyclonal antibody (P-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-PDGFRβ mAb (clone 42F9, Cell Signaling Technology), and anti-α-tubulin mAb (clone YL1/2, AbD Serotec, Kidlington, UK). The LAS-3000 mini system (Fujifilm, Tokyo, Japan) was used for visualization and quantification of signals.