Hrp linked anti rabbit igg secondary antibody

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Life Technologies (Carlsbad, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IFN-γ, and IL-2 were from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from USB Corp. (Cleveland, OH, USA), and the LDH release detection kit was obtained from Roche Applied Science (Indianapolis, IN, USA). All kits were used according to the manufacturers’ protocols. CTX monohydrate, LPS, concanavalin A (ConA), and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). YAC-1 cells, a mouse lymphoma cell line, and RAW264.7 cells, a mouse macrophage cell line, were obtained from ATCC (Manassas, VA, USA; nos. ATCC^®TIB-160^TM and ATCC^®TIB-71 ^TM). Antibodies against β-actin, iNOS, p-NF-κB p65, NF-κB p65, p-IκB, IκB, p-Akt, Akt, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, and p38, and HRP-linked anti-rabbit IgG secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of the highest grade commercially available.

Nuclear extracts were prepared using the Nuclear Complex Co‐IP Kit according to the manufacturer's instructions (Active Motif #54001). Protein extracts were separated by electrophoresis on 4‐20% Mini‐PROTEAN^® TGX™ gels (Bio‐Rad #4561096) in TGS (25 mM Tris, 190 mM glycine, 0.1% SDS) before transfer to nitrocellulose membranes using a Trans‐Blot^® Turbo™ Transfer system (Bio‐Rad). Membranes were blocked with 5% (w/v) milk powder in TBS‐Tween (TBST) (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween) at room temperature for 1 h before incubation overnight at 4°C with 1:1,000 dilution of primary antibodies in 5% (w/v) milk powder in TBST. Primary antibodies: JUND (Thermo Scientific #720035), p‐STAT5 (Y694) (Cell signaling #9351S) STAT5 (Santa Cruz #sc‐835X), RUNX1 (Abcam #ab23980), B2M (Abcam #ab75853), and ETS1 (Santa Cruz #sc‐350X). Membranes were washed with TBST and incubated with 1:10,000 dilution of HRP linked anti‐rabbit IgG secondary antibody (Santa Cruz #sc‐2054) for 1 h at room temperature. Membranes were visualized using the SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific #34577).