Protein Extraction and Western Blot Analysis

Protein extracts were prepared from flash-frozen tissue after homogenization in RIPA lysis buffer (Incomplete RIPA buffer, 7× protease inhibitor cocktail, 200mM sodium orthovandate, 1M sodium fluoride, 100mM PMSF) as previously described [15 (link)]. Total proteins (30–50 µg) were subjected to Western blot analysis with the following antibodies; anti-pMek1/2, anti-Mek1/2, anti-pErk1/2, anti-Erk1/2, anti-pp90Rsk, anti-pRsk1/2, anti-pStat3, and anti-Stat3 (all rabbit, Cell Signaling Technology, MA, USA; 1:1000 concentration). Mouse anti-vinculin (Santa Cruz, CA, USA; 1:2000) and mouse anti-β-actin (Sigma Aldrich, Missouri, USA; 1:10,000) were used as loading controls. Furthermore, membranes were washed and incubated with anti-rabbit or anti-mouse (Invitrogen, CA, USA; 1:5000) horseradish peroxidase-conjugated antibody. Western blot images were acquired using EMD Millipore Luminata HRP chemiluminescence substrate (Millipore, MA, USA) and signal acquired in Bio-Rad chemiluminescence detector (Bio-Rad Laboratories, CA, USA). The signals were semiquantified using image J (ImageJ; www://rsb.info.nih.gov/ij/).