Zfp91

Cells were seeded (OVCAR-3: 500 × 10³, MIA PaCa-2: 200 × 10³, BxPC-3: 500 × 10³, MCF-7: 300 × 10³ cells/well) in 6-well plates. Following treatment, cells were lysed at 4 °C by sonication with lysis buffer (25 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl, 17 mM Triton X-100, 3.5 mM SDS, pH 7.4) supplemented with protease inhibitor and phosphatase inhibitor. The cells were then spun at 12,000 rpm at 4 °C for 10 min and collected for determining the protein concentration with the BCA assay (ThermoFischer Scientific, Waltham, MO). Samples were then prepared and loaded onto 10 or 12 % acrylamide (BioRad, Hercules, CA) gels followed by the transfer of the proteins onto PVDF membranes (EMD Millipore, La Jolla, CA). The membranes were blocked for 1 h prior to incubation with the primary antibodies using Odyssey blocking buffer (LI-COR Biosciences). Membranes were then probed for STAT3/pSTAT3/STAT1/NQO1 (Cell Signaling, Danvers, MA, 1:1000), GAPDH (Cell Signaling, Danvers, MA, 1:4000), GSPT (Cell signaling, 1:1000), ZFP91 (Bethyl, 1:1000). Following overnight incubation with the primary antibodies at 4 °C, the membranes were incubated with the secondary antibodies (anti-rabbit, Cell Signaling, 1:7500 or anti-mouse, Cell Signaling, 1:5000) for 1 h and imaged with the Odyssey imaging system (LI-COR Biosciences).