Murine anti phospho stat3 py705 and anti stat3 mabs

Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Brij97) containing 2 mM Na Orthovanadate and protease inhibitors (Roche Diagnostics, Complete Mini 04693124001). Lysates were resolved under reducing conditions by SDS-PAGE (10% or 13% acrylamide) and analyzed by Western blotting using the following antibodies: rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera (Cell Signaling Technology, 9167 and 9172, respectively), murine anti-phospho-STAT3 (pY705) and anti-STAT3 mAbs (BD Transduction Laboratories, 612,356 and 610,190, respectively), rabbit anti-SOCS3 (Cell Signaling Technology 2932) and murine α-tubulin or β-actin mAbs (Sigma-Aldrich T6074 and A2228, respectively). Proteins were detected by ECL Prime (GE Healthcare, RPN2232) and visualized by a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge).

Cells, detached by 2 mM EDTA solution in PBS, were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Brij97) containing 2 mM Na Orthovanadate and protease inhibitors (Roche Diagnostics, Complete Mini 04693124001). Lysates were resolved under reducing conditions by 10% SDS-PAGE and analyzed by Western blotting using the following antibodies: anti-IDO murine mAb (Upstate, clone 10.1, 05-840), rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera (Cell Signaling Technology, 9167 and 9172, respectively), murine anti-phospho-STAT3 (pY705) and anti-STAT3 mAbs (BD Transduction Laboratories, 612356 and 610190, respectively), and anti-β-actin or α-tubulin mAbs (Sigma-Aldrich, A2228 and T6074, respectively). Proteins were detected by ECL Prime (GE Healthcare, RPN2232) and autoradiography.