T her2

Whole cell lysates were harvested using the PRO-PREPTM Protein Extraction Solution (iNtRON, Sungnam, Korea). Western blot analysis was performed as described previously [21 (link),22 (link)]. Blots were incubated with anti-PDGFRA (sc-398206, 1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), PDGFRB (sc-374573, 1:1000, Santa Cruz), p-STAT3 (ab76315, 1:100000, Abcam), p-ERK (sc-7383, 1:1000, Santa Cruz), t-STAT3 (sc-8019, 1:1000, Santa Cruz), t-ERK (#9102, 1:1000, Cell signaling technology, Danvers, MA, USA), t-HER2 (sc-33684, 1:5000, Santa Cruz), or β-actin (LF-PA0207, 1:5000, AbFrontier Co. Ltd., Seoul) antibodies in 1% Tris-buffered saline with 0.01% Tween-20 (TBST) at 4 °C overnight (O/N). Blots were washed 3–4 times in TBST and incubated with appropriate secondary antibodies in TBST buffer for 1 h at room temperature. Blots were washed 3–4 times with TBST buffer and visualized with the ECL™ Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA).

For immunoblotting, samples were lysed on ice using lysis buffer and boiled for 5 min in Laemmli sample buffer. Samples were loaded into SDS-polyacrylamide gels under denatured conditions and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p-EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p-HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (CST) at RT for 1 h. Protein signals were developed using ECL^TM prime reagent (GW Healthcare, Bucks, UK).