Western Blot Analysis of Signaling Proteins

Cell lysates were prepared and western blot analysis was performed as described previously [22 (link)]. Antibodies against the following proteins were used: phosphorylated EGFR (p-EGFR,Y1068, #2234), Total EGFR (T-EGFR, #2232),phosphorylated Signal transducer and activator of transcription 3(p-STAT3,Y705, #9145), Total STAT3 (T-STAT3, #4904), phosphorylated v-Akt Murine Thymoma Viral Oncogene (p-AKT, Ser473, #9271), Total AKT (T-AKT, #9272), phosphorylated Extracellular Signal-regulated Kinase (p-ERK, Thr202/Tyr204, #9101), Total ERK (T-ERK, #9102), heat shock protein 60(HSP 60, #12165), Vimentin(#5741), phosphorylated focal adhesion kinase (p-FAK, Y397, #3283), Total FAK(T-FAK, #3285), α-tubulin(#2144) (all antibodies except type I collagen were purchased from Cell Signalling Technology, Danvers, MA, USA, 1:1000). type I collagen (abcam, Cambridge, UK, #34710). The band densitometric analysis was performed by using the public software Image J (Wayne Rasband National Institutes of Health, Bethesda, MD, USA). Expression of α-tubulin was used to correct for variation in sample loading.