Ecl system

For immunoblotting analysis, lung samples and cell lysates lysed by RIPA buffer (Beyotime, China) were resolved by SDS–PAGE and transferred to the polyvinylidene fluoride membrane (Millipore, United States). Immunoblots were visualized by the ECL system (Fdbio Science, China). The following antibodies were used in immunoblotting analysis: antibodies for anti-IFITM3 (1:2,000), anti-MFN2 (1:2,000), anti-IFN-β (1:500) antibody and anti-IL-1β (1:1,000) were from Abcam. β-actin antibody (1:2,000) was purchased from Fude Biotechnology. Anti-phospho-IRF3 (1:1,000), and anti-TNF-α (1:2,000) antibodies were purchased from Cell Signaling Technology. Anti-MAVS (1:500) antibodies were obtained from Proteintech Group. β-actin was used as an internal control and the relative densities of the measured protein were quantified by image J software.

Adipose tissues were lysed with RIPA buffer (added with 1 mM PMSF) and then centrifuged at 13,000 rpm for 15 min at 4°C. Protein concentration was measured with pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Lysates were resolved by 10% SDS-PAGE, transferred to PVDF membrane (Millipore) and probed with indicated antibodies. The bound antibodies ware detected by second HRP-conjugated antibodies for 1 h at room temperature and visualized by ECL system (Fdbio science, Hangzhou, China).

For western blotting analysis, the proteins of lung samples and cell lysates lysed by RIPA buffer (Beyotime, Shanghai, China) were separated using SDS-polyacrylamide gel electrophoresis. Subsequently, the proteins resolved in the gel were transferred to the polyvinylidene fluoride membrane (Millipore, MA, USA). Western blots were visualized using the ECL system (Fdbio Science, Hangzhou, China) and Quantity One software (Bio-Rad, USA) was used to analyze the intensity of individual bands. Data were normalized with β-actin as a loading control in each individual sample. The following antibodies were used in western blotting analysis: influenza virus NP rabbit pAb (1:2000), IFITM3 rabbit pAb (1:2000), IL-1β rabbit pAb (1:1200) and IL-6 rabbit pAb (1:1200) were provided by Abcam (MA, USA). Mfn2 mouse mAb (1:2,000) and IL-10 mouse mAb (1:2000) were obtained from Santa Cruz Biotechnology (CA, USA). β-actin rabbit pAb (1:2000) was from Bioworld (MN, USA). IFN-β rabbit pAb (1:500) was from OriGene Technology (MD, USA). p-IRF3 rabbit mAb (1:1000), NF-κB p65 rabbit mAb (1:2000) and TNF-α rabbit pAb (1:2000) were provided by Cell Signaling Technology (MA, USA). MAVS rabbit pAb (1:500) and STING rabbit pAb (1:500) antibodies were obtained from Proteintech Group (IL, USA).