Mp autoradiography films

For Western blot analysis, whole-cell extracts were harvested using RIPA lysis buffer (strong) (CWBIO, Beijing, China) containing protease inhibitor cocktail (CWBIO, Beijing, China) and phosphatase inhibitor cocktail (CWBIO, Beijing, China) at 30 min, 60 min, and 120 min postinfection, separated by SDS–PAGE and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). Subsequently, the membranes were incubated with primary antibodies against monoclonal mouse anti-β-actin (1:4000, TransGen, China), rabbit anti-p-p44/42 MAPK (1:1000, CST, USA), rabbit anti-p-SAPK/JNK (1:1000, CST, USA) or rabbit anti-p-p38 (1:1000, CST, USA) overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:1000, Beyotime, China) or HRP-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, China). Finally, the bands were visualized using Amersham^® Hyperfilm^® ECL™ and MP Autoradiography Films (GE Healthcare).

HEK293T cells were lysed in 250 μL NP40 lysis buffer (50 mM Tris‐HCl; pH 7.3, 150 mM NaCl, and 1% (v/v) NP40) along with freshly added protease and phosphatase inhibitors (Roche). Lysates were incubated at 4°C overnight with anti‐Flag antibody conjugated to agarose beads (Sigma-Aldrich) or anti-HA antibody conjugated to agarose beads. The beads were washed with lysis buffer and analysed by western blotting. Primary antibodies used were polyclonal anti‐Flag (Sigma-Aldrich), anti‐HA (Zymed), anti‐ERK1/2, anti-IRF3, or anti-TBK1 (Cell Signaling Technology). Donkey anti-rabbit HRP-conjugated antibody (Amersham biosciences) was used as the secondary antibody.
Cell lysates were prepared as previously described [51 (link)], resolved in a 10% or 12% Tris/Glycine gel (10% or 12% acrylamide, 1.5 M Tris (pH 8.8), 10% SDS, 10% APS, Tetramethylethylenediamine), and were subjected to western blot analysis with anti-pERK, anti-pp38 and anti-pJNK antibodies (Cell Signaling Technology) for MAP kinases activation, anti-pIRF3 (Ser396) (Cell Signaling Technology) for IRF3 phosphorylation, or anti-pTBK1, anti-TBK1 or anti-IKKε for their phosphorylation and expression, respectively. The blots were exposed to Amersham^® Hyperfilm® ECL™ and MP Autoradiography Films (GE Healthcare). The intensity of the specific bands was quantified using ImageJ software.

Proteins were harvested using protein lysis buffer (1 M Tris–Cl, 5 M NaCl, 100% NP-40) containing 1× Halt Protease and Phosphatase Inhibitor Cocktail and 0.5 M EDTA (Thermo scientific). Protein samples were subjected to SDS-PAGE and Western blot was performed using the following antibodies: anti-DUSP12 (Santa Cruz, 1:500 dilution), anti- beta actin (Cell signaling, 1:1,000 dilution), anti-ERK (Cell Signaling, 1:1,000 dilution), antiphosphorylated-ERK(Sigma, 1:1,000 dilution), anti-JNK(Cell Signaling, 1:1,000 dilution), antiphosphorylated-JNK (Sigma, 1:1,000 dilution), anti-p38 (Cell Signaling, 1:1,000 dilution), or antiphosphorlated-p38 (Sigma, 1:1,000 dilution). Primary antibodies were detected with either antigoat (Santa Cruz, 1:4,000 dilution) or anti-rabbit IgG (Cell Signaling, 1:4,000 dilution) horseradish peroxidase-coupled secondary antibodies. Images were captured using Amersham^® Hyperfilm^® ECL™ and MP Autoradiography Films (GE Healthcare).