Horseradish peroxidase conjugated anti mouse 7076s

B and T cells were isolated from wild-type spleens using magnetic beads as above. Cell lysates were prepared from 3 × 10⁶ cells by incubating with lysis buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF) and denatured in Laemmli buffer (Bio-Rad, Inc.) with 355 mM 2-mercaptoethanol. After boiling, proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and immunoblotted with mouse monoclonal antibody to Top2b (1 μg/mL, ThermoFisher Scientific, MA5-24310) followed by horseradish-peroxidase conjugated anti-mouse (7076S, Cell Signaling Technology, Inc.), for detection by chemiluminescence (ThermoFisher Scientific). Anti-GAPDH-HRP was used to assess gel loading (1:3000, Proteintech, HRP-60004).

Sulforaphane (≥95% HPLC), hemin, actinomycin, angiotensin II, N-acetylcysteine (NAC), dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and β-actin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). NG-nitro-L-arginine methyl ester (L-NAME), LY294002 (PI3K inhibitor), pyrazolopyrimidines (PP2, an Src kinase inhibitor) were bought from Calbiochem (La Jolla, CA, USA). Primary antibodies for eNOS (32027S), phospho-eNOS (Ser 1177) (9570S), Akt (4691s), phospho-Akt (Ser 473) (4060S), and phospho-Src (Tyr 416), (6943S) as well as horseradish peroxidase-conjugated anti-mouse (7076S) or anti-rabbit IgG (7074S) antibodies were obtained from Cell Signaling Technology (Beverly, MA). All other chemicals, unless otherwise noted, were pure, analytical grade. The cell culture media and reagents involved were obtained from Invitrogen (Carlsbad, CA, USA).