Nf κb p65

The lysates of treated cells were reconstituted with loading buffer and run by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Nitrocellulose membranes (Millipore, USA) containing the transferred proteins were soaked in blocking buffer for 2 h and then separately incubated at 4°C overnight with the following primary antibodies: SelS (Sigma, USA), endothelial nitric oxide synthase (eNOS; Proteintech, Wuhan, China), p-c-jun (Proteintech, Wuhan, China), c-jun (Proteintech, Wuhan, China), p-p38 MAPK (Abcam, USA), p38 MAPK (Abcam, USA), inhibitory kappa B α kinase β (IKKβ, CST, USA), p-IKKβ (CST, USA), inhibitory kappa B α (IκBα, CST, USA), p-IκBα (CST, USA), NF-κB p65 (Bioworld, USA), Lamin B (Proteintech, Wuhan, China), and GAPDH (Proteintech, Wuhan, China). After washing for 30 min, the Nitrocellulose membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were subsequently treated with enhanced chemiluminescence (ECL, Thermo Scientific, USA) to develop the protein bands, and images were captured using the ChemiDoc MP Imaging System (Bio-Rad, USA). Quantitative analysis of the band intensities was performed with the Quantity One 4.52 software program (Bio-Rad, USA). Lamin B and GAPDH were used as internal controls.

The cells were lysed in Cell Lysis Buffer for Western and IP (Beyotime) and incubated at 4 °C for 15 min. Protein extracts were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes before overnight incubation with primary antibodies directed against Asb2 (Santa Cruz), IκBα (Cell Signaling Technology, 1:600 dilution), NF-κB-p65 (BioWorld), Caspase 3 (Proteintech Group, 1:2000 dilution), NICD (abcam), Hes1 (abcam), lamin (Wuhan Boster Biological Technology) or GAPDH (Wuhan Boster Biological Technology) at 4 °C. The membrane was washed with 0.1% Tween-20 in Tris-buffered saline and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG secondary antibody (Wuhan Boster Biological Technology) for 1 h at room temperature. The immunoreactive bands were visualized using an ECL Western blotting detection kit (Thermo, Waltham, MA, USA) with light-sensitive film.

An M-MLV reverse transcription kit (Takara Bio, Inc., Shiga, Japan), TaqE (Takara), dNTP (Takara), DNA marker (Takara), SYBR-Green mix (Bio-Rad, Hercules, CA, USA), agarose (Promega Corporation, Madison, WI, USA), diethylpyrocarbonate (Sigma, St. Louis, MO, USA) and TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) were used in this study. Primers were synthesized by Sangon Co., Ltd. (Shanghai, China) as follows: GAPDH (201 bp) forward, 5′-CTCATGACCACAGTCCATGC-3′ and reverse, 5′-CACATTGGGGGTAGGAACAC-3′; TLR7 (117 bp) forward, 5′-ACGCTTTCTTTGCAACTGTG-3′ and reverse, 5′-TTTGTGTGCTCCTGGACCTA-3′; and MYD88 (136 bp) forward, 5′-TGGTGGTTGTTTCTGACGAT-3′ and reverse, 5′-GGAAAGTCCTTCTTCATCGC-3′. NF-κB-P65 and p-c-jun rabbit monoclonal primary antibodies were purchased from Bioworld Technology Co., Ltd (Nanjing, China), while IgG goat polyclonal secondary antibody was purchased from the Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China) and cell lysis buffer was bought from Beyotime (Nanjing, China). The western blot analysis was conducted at the Key Laboratory of Antiviru of the Ministry of Education. Enzyme-linked immunosorbent assay (ELISA) kits, IL-1β, TNF-α and IL-6 were obtained from Bender MedSystems (Vienna, Austria).