Peroxidase affinipure goat anti mouse igg h l antibody

Mice were perfused with ice-cold Dulbecco’s PBS (-) and the brain was dissected and lysed in lysis buffer (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% [vol/vol] Nonidet P40 Substitute, 0.5 mM EDTA) plus EDTA-free protease inhibitor cocktail cOmplete (Roche) and phosphatase inhibitor phosSTOP (Roche). Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Mini-Protean TGX Precastgels 4–20%, Bio-Rad, Richmond, CA), and transferred to polyvinyldifluoride (PVDF) membranes using the Trans-Blot Turbo Transfer system (Bio-Rad, Richmond, CA, USA). The membranes were blocked in 5% skim milk in PBST (0.05% Tween 20 in PBS) for 1 h and probed with the primary antibodies overnight and secondary antibodies for 1 h at RT. The following antibodies were used for Western blotting: Peroxidase AffiniPure goat anti-mouse IgG (H + L) antibody (1:10,000, 115–035-146, Jackson) and anti-GAPDH antibody (1:1000, sc32233, Santa Cruz Biotechnology). Bands were detected using Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare) and images were captured using LAS-4000 mini.

Following antibodies were used in this study: anti-p-Smad (CST, 13820S), anti-Smad (ProteinTech, 10429-1-AP), anti-p-STAT3 (ser727; CST, 9134S), anti-STAT3 (CST, 9139S), anti-SOCS3 (CST, 2923S), anti-myc (CST, 2276S), anti-β-actin (Santa Cruz, sc-1616), Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) Antibody (Jackson ImmunoResearch Labs, 115-035-003), Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) Antibody (Jackson ImmunoResearch Labs, 111-035-003). For CO-IP experiment, the following agents and antibodies were used, Protein G PLUS-Agarose Antibody (Santa Cruz, sc-2002), Normal Mouse IgG Antibody (Santa Cruz, sc-2025), anti-myc (CST, 2276S), Peroxidase-AffiniPure Goat Anti-Mouse IgG, Light Chain Specific Antibody (Jackson ImmunoResearch Labs, 115-035-174).