Genegnome bio imaging system

Cells were lysed in RIPA buffer plus protease inhibitors (Millipore, USA). The lysates were then subjected to SDS-PAGE, electrotransferred to PVDF membranes (Millipore, USA), blocked with 5% nonfat milk, incubated with antibodies for 2 hours at room temperature, including anti-beta-tubulin (Abmart, M20005, 1:2000, China), anti-BANF1 (Santa Cruz, sc-33787, 1:200, USA), HRP anti-rabbit (KPL, 4751-1516, 1:2000, USA) and HRP anti-mouse (KPL, 0751-1809, 1:2000, USA), and visualized using cECL Detection Reagents (CWBIO, China). Images were acquired using the Gene Gnome Bio Imaging system (Syngene, UK).

Western blotting procedures were previously described [12 (link), 13 (link)]. Briefly, protein samples were separated by SDS-PAGE on a 10% gel, soaked in transfer buffer, and transferred to a PVDF membrane. The membrane was incubated with primary Abs overnight then with HRP-conjugated secondary Abs for 60 min at room temperature. Protein bands were visualized with an ECL Prime kit (GE Healthcare UK, Ltd.), and immunoblotting signals were acquired with a GENEGNOME Bio Imaging System (SYNGENE).

Western blot was used to determine the protein levels of TLR4, as described previously 15. Heart tissue was homogenized and isolated cardiomyocytes were lysed in RIPA buffer supplemented with protease inhibitors (Beyotime Institute of Biotechnology, Jiangsu, China), sonicated on ice and protein concentration was determined using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). The lysates (20 μg of total proteins) were electrophoresed on a 10% SDS‐PAGE gel and transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively. The signal was visualized using chemiluminescence reagents, scanned with a GeneGnome Syngene Bio Imaging system and quantified by densitometry.

Total proteins were extracted from fresh tissues and cells using RIPA Protein Lysis solution (Pierce, IL, USA) and quantified by the Bradford method. Prepared samples were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a polyvinylidene fluoride membrane (Millipore, MA, USA) using the Tetra Handcast system (Bio-Rad, USA). The membrane was blocked for 1 h at room temperature or overnight at 4 °C in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. Then it was incubated overnight at 4 °C with the appropriate primary antibody, and followed by the secondary antibody for 70 min at room temperature. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (Syngene, UK) with an electrochemiluminescence kit (Pierce, IL, USA).
The primary anti-SOX3 antibody was purchased from Abcam (MA, USA), and the anti-β actin antibody was purchased from Santa Cruz Biotechnology Inc. (CA, USA). Other primary antibodies (anti-E-cadherin, anti-CK-18, anti-N-cadherin, anti-vimentin, anti-Snail1, anti-Twist1, anti-Slug, anti-ZEB1, anti- ZEB2, anti-FLAG) were purchased from Cell Signaling Technology (MA, USA). Anti-mouse or anti-rabbit HRP-conjugated secondary antibodies were purchase from Santa Cruz Biotechnology Inc. (CA, USA).