Hrp horseradish peroxidase labeled goat anti mouse igg

Cells were lysed in a buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0 for 1 h. The supernatant was collected after centrifugation, and 6 μg of protein was used for Western blotting. Briefly, the proteins were separated on NuPage 4–12% Bis-Tris gels (Invitrogen) by electrophoresis and transferred to nitrocellulose i-Blot gel transfer stacks (Invitrogen). The membrane was washed with PBS with 0.1% Tween 20 (PBST), placed in blocking buffer (PBST containing 5% nonfat milk) for 1 h. Then the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκB-α) (Cell Signaling Technology, Danvers, MA; 1:1,000), phosphorylation interferon regulatory factor 3 (pIRF3) (Cell Signaling Technology; 1:1,000), and mouse monoclonal anti-β-actin (Sigma; 1:5,000) as an internal control. After washing with PBST, membranes were incubated with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Cell Signaling Technology; 1:5,000) or goat anti-rabbit IgG (Cell Signaling Technology; 1:1,000) as secondary antibodies. Specific proteins were visualized using Immunostar LD reagent (Wako Pure Chemical, Osaka, Japan), and the chemiluminescence was detected using the C-DiGit blot scanner (LI-COR).