Pmypt 1 thr 850

Lysates were prepared by homogenizing the flash-frozen tissues in PBS supplemented with a protease inhibitor cocktail. Total protein content was determined by using Bradford’s reagent (Biorad, Hercules, CA). Then, an equal amount of protein was denatured with beta-mercaptoethanol for Western blot analysis. Protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the segregated protein was transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked using fat-free milk and incubated overnight at 4 °C with primary antibodies specific to pMYPT-1 (Thr-850) (EMD Millipore, Billerica, MA; 1:700), pSTAT-3 (Y705) (Abcam, Cambridge, MA; 1:1000), and actin (Sigma-Aldrich, St. Louis, MO; 1:10,000). Next day, PVDF membranes were washed with TTBS followed by incubation with antirabbit and antimouse secondary antibodies (1:2000 in fat free milk), respectively. Membranes were developed using chemiluminescence detection reagent (Bio-Rad, Hercules, CA). The blots were quantitated with UN-SCAN-IT graph digitizing software (Silk Scientific, Orem, UT) using Actin as an internal control.