Protein Phosphorylation Analysis in Lung Cancer

Western blotting was performed to assess protein phosphorylation and expression levels in lung cancer cells treated with AC-93253 iodide as described previously [20 (link)]. The EGFR, STAT3 (F-2), PI3K, phospho-MEK1/2 (Ser 218/Ser 222), MEK, phospho-ERK (Tyr204), ERK, Paxillin, and p130cas were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phospho-Src (pY418), phospho-FAK (Tyr576), and FAK were purchased from Invitrogen (Carlsbad, CA, USA). Phospho-EGFR (Tyr1068), phospho-STAT3 (Tyr 705), phospho-PI3K (Tyr458), phospho-SAPK/Jun N-terminal kinase (JNK) (Thr183/Tyr185), SAPK/JNK, phospho-Paxillin (Tyr118), and phospho-p130cas (Tyr410) were purchased from Cell Signaling Technology (Beverly, MA, USA), and the primary antibody to Src was produced in our laboratory (ATCC CRL-2651). GAPDH (Upstate Biotechnology, Lake Placid, NY, USA) was used as a loading control. The mRNA expression levels of Src and related genes were detected using a real-time PCR machine (ABI prism 7300 Sequence Detection System, Applied Biosystems, Carlsbad, CA, USA) and SYBR Green Reagent (Roche, Basel, Switzerland). TATA-box binding protein (TBP) was used as an internal control (GenBank X54993). Details regarding the procedures and calculations used to perform the experiments are provided elsewhere [21 (link)].