Hrp labeled goat anti mouse secondary antibody

The transfected cells were added to RIPA lysate and protease inhibitor to extract total protein (EpiZyme, Shanghai, China). After lysis on ice for 30 min, the proteins were centrifuged at 12000 rpm for 15 min at 4°C. The loading volume of each sample was measured using a BCA kit (GenStar, Beijing, China). Identical masses of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to PVDF membranes (Bio-Rad, UK) and sealed with skim milk powder. PVDF membranes were incubated overnight at 4°C with primary antibodies against VCAM-1 (1:500, Proteintech, USA), ICAM-1 (1:2500, Proteintech, USA), CDH2 (1:1500, Proteintech, USA), and GAPDH (1:10000, Proteintech, USA). Finally, the HRP-labeled goat anti-mouse secondary antibody (1:5000, Proteintech, USA) was incubated with the filter membrane for 1 h. GNG12 protein expression levels were detected using an imager and a chemiluminescent substrate (ECL) kit (Thermo Fisher Scientific, USA).

HEK293T cells seeded in 10-cm plates (1 × 10⁷ cells/well) were co-transfected with 5 μg FLAG-tagged LcMFN2 and 5 μg His-tagged LcMAVS or empty vector. After 24 h, cells were lysed with prechilled lysis buffer (50 mmol/L Tris HCL, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-X-100, and 1 mmol/L PMSF). Proteins were precipitated using BeyoMag™ Anti-Flag Magnetic Beads (Beyotime) at 4 °C for 6 h. Following three times washing with TBS/T, the immunocomplex was eluted with 3 X Flag Peptide (sigma). For immunoblotting, denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electro-imprinted on 0.2 μmol/L PVDF membrane (Millipore, Bedford, MA) using a micro-trans-Blot cell system (Bio-Rad), and then blocked with Quick block blocking buffer (Beyotime). The membrane was incubated overnight at 4 °C with primary antibodies against FLAG-Tag (MBL, M185-3L, 1:10,000 dilution in TBS/T containing 1% non-fat dry milk) or His-Tag (1:3000 dilution in TBS/T containing 1% non-fat dry milk), followed by washing three times with TBS/T. Then, the membrane was probed with the HRP-labeled goat anti-mouse secondary antibody (Proteintech, SA0001-1, 1:10,000). Signals were detected using an ECL chemiluminescent system captured by a gel imaging system (GE Healthcare).