Mouse anti flag mab

Protein samples were separated by 12% SDS-PAGE and were transferred onto polyvinylidene difluoride (PVDF) membranes (catalog no. ISEQ00010; Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h, followed by incubation with indicated primary antibodies at room temperature for 2 h; for example, rabbit anti-TRAF5 polyclonal antibody (pAb) (catalog no. orb186283; Biorbyt, Cambridgeshire, UK), rabbit anti-MAPK14 (p38 MAPK) pAb (catalog no. CSB-PA003661; Cusabio, Wuhan, China), rabbit anti-phospho-MAPK14 (T180/Y182) pAb (catalog no. CSB-PA000647; Cusabio, China), mouse anti-GFP monoclonal antibody (mAb) (catalog no. KM8009; SUNGENE BIOTECH, Tianjin, China), mouse anti-Flag mAb (catalog no. CW0287; CWBIO, Beijing, China), and mouse anti-GST mAb (catalog no. CW0291; CWBIO, China). After incubation with horseradish peroxidase-conjugated goat anti-rabbit (or mouse) IgG secondary antibody (catalog no. LK2001 or LK2003; SUNGENE BIOTECH, China), the signal was visualized using an image analysis system (Bio-Rad, Hercules, CA, USA).

Cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer with protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and Halt™ phosphatase inhibitor cocktail (Roche, Switzerland). The protein concentration was determined with BCA Protein Assay Kit. The samples were separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies used include rabbit anti-TRAF6 polyclonal antibody (pAb), rabbit anti-NF-κB p65 pAb, rabbit anti-phospho-NF-κB p65 pAb, rabbit anti-Flag pAb (SIGMA, USA), mouse anti-GST monoclonal antibody (mAb), mouse anti-GFP mAb and mouse anti-Flag mAb (CWBIO, China). After five washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (or mouse) IgG (Vazyme, China) secondary antibody for 2 h at room temperature. After another five washes, the signal was detected using an image analysis system (Bio-Rad, USA). The secreted protein levels of IFN-β and IL-6 in cell culture supernatant were determined with IFN-β antibody (LSBio) and IL-6 antibody (R & D Systems), respectively. All samples were in triplicate and read at 450 nm using a Multiskan FC Microplate Photometer (Thermo, USA).