Rabbit anti stat4

Total protein was extracted from the cells with a lysis buffer containing 1% NP-40, 20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM EDTA, and 50 mM NaF, supplemented with 100 mM orthovanadate, 200 mM PMSF, 1% aprotinin, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml antipain. Protein fractions were separated by SDS-PAGE and electrotransferred onto PVDF membranes. The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]). Ab binding was detected by incubation with HRP-conjugated anti–mouse or anti–rabbit secondary Abs (GE Healthcare), with the ECL system (Thermo Fisher Scientific).

Western blot analysis was performed as described in Fortunato et al.22 using the after antibodies: rabbit anti‐STAT4 (1:1000, Cell Signaling), mouse anti‐actin (1:2000, Sigma Aldrich) and goat anti‐rabbit or goat anti‐mouse secondary antibodies conjugated to horseradish peroxidase (1:5000, GE Healthcare). Signal detection was performed via chemiluminescence reaction (ECL, GE Healthcare) using MINI HD9 Western Blot Imaging System (Cleaver Scientific Ltd, United Kingdom). WB quantification was performed using ImageJ software analysis.