Total stat1 t stat1

Protein samples (10 µg) from HFL-1 were resolved on NuPage Novex 4–12% Bis–Tris gel (Thermo Fisher Scientific, MA) electrophoresis, transferred, and immunoprobed with mouse monoclonal antibody for p-STAT1, total STAT1 (t-STAT1) (1:1000 and 1:500 respectively, Cell Signaling Technologies Inc. MA). Alkaline phosphatase conjugated secondary antibody (Thermo Fisher Scientific) was followed by Chemiluminescent detection (ChemiDoc XRD-J Bio-Rad Laboratories, Inc., CA). Densitometry was performed using the Quantity One (Bio-Rad) and p-STAT1 indexed to t-STAT1.

Tumor cell pellets were sonified in HIF-lysis buffer (6.65 M Urea, 10% glycerol, 1% SDS, 10 mM Tris; pH 7.4), 100 ng protein per sample was loaded on SDS polyacrylamid gels together with SDS loading buffer (0.5 M Tris, pH 6.8; 2% SDS, 20% glycerol, 0.002% bromphenol blue, 5 mM DTT). Proteins were blotted on a nitrocellulose membrane, incubated with β-actin (Sigma-Aldrich), phospho-STAT (pSTAT1) (Cell Signaling), total STAT1 (tSTAT1) (Cell Signaling), pSTAT3 (Cell Signaling), tSTAT3 (Cell Signaling), and visualized by IRDye 680- and IRDye 800-coupled secondary Abs using the Li-Cor Odyssey imaging system (LICOR Biosciences, Bad Homburg, Germany).