Imager 800

The MinuteTM Total Protein Extraction kit (Inventbiotech, Plymouth, MN, USA) was used to extract total proteins. Briefly, 10% SDS-PAGE was used to separate equal proteins, which were subsequently electroblotted to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the primary antibodies, including TIPE2 (1:500, Proteintech, China), p38, p65, p p65 (1:1000; Cell Signaling Technology), and β-actin (1:500, Servicebio), were added and incubated overnight at 4°C. Horseradish peroxidase-goat anti-rabbit IgG (1:10000; CWBiotech, Beijing, China) was incubated at room temperature for 1 h. Finally, the blots were visualized with an Amersham Imager 800 instrument.

Cardiac tissue or cells were lysed with RIPA lysis buffer. The prepared protein samples were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane (Millipore, USA). 5% nonfat milk was used for blocking at room temperature for 1 h, and then the membrane was incubated with 1 antibody at 4 °C overnight. The next day, incubation with secondary antibodies was performed for 1 h at room temperature before enhanced chemiluminescence using an Amersham Imager 800 (Additional file 1).