Rabbit anti phospho stat1 py701 and anti stat1 anti sera

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.

Cells were lysed with 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Brij97, 2 mM Na Orthovanadate, and protease inhibitors (Roche Diagnostics, Complete Mini 04693124001). Proteins were resolved by 10% SDS-PAGE under reducing conditions, and Western blotting was carried out according to standard techniques, with rabbit anti-phospho-STAT1 (pY701) and anti-STAT1 anti-sera (Cell Signaling Technology, 9167 and 9172, respectively), mouse anti-phospho-STAT3 (pY705) and anti-STAT3 mAbs (BD Transduction Laboratories, 612,356 and 610,190, respectively) and α-tubulin mAb (Sigma-Aldrich, T6074).
Immunoreactive proteins were detected by ECL Prime (GE Healthcare, RPN2232) and a chemiluminescence gel documentation and analysis system (MINI HD, UVITEC, Cambridge, UK). Densitometric analyses of relevant bands and whole Western blot images are shown in the Supplementary Materials Figure S4.