Anti bhlhe40

Total protein was extracted from rat embryonic spinal cords (E11 and E12) or C17.2 cells using radioimmunoprecipitation (RIPA) buffer (Solarbio Science & Technology, Beijing, China). Protein concentrations were determined by a bicinchoninic acid assay (Solarbio Science & Technology). Proteins were separated by 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk solution, the membranes were incubated with primary antibodies at 4°C overnight. Following primary antibodies were applied in the experiment: anti-LC3 (Abcam, Cambridge, MA, United States; 1:1000), anti-LAMP2 (ProteinTech, Chicago, IL, United States; 1:1000), anti-Sirt1 (Cell Signaling Technology, Boston, MA, United States; 1:1000), anti-Bhlhe40 (ProteinTech, 1:1000), anti-PINK1 (ProteinTech, 1:1000), anti-VDAC1 (Abcam, 1:2000) and anti-GAPDH (ProteinTech, 1:5000). On the following day, the membranes were washed, and incubated with corresponding secondary antibodies (ProteinTech; 1:5000) at room temperature for 1.5 h. Enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, United States) was applied to detect protein-antibody interactions, which were visualized using a chemiluminescence detection system (C300, Azure Biosystems, United States).

Atrial samples from the patients with AF or mice were removed and fixed in 4% paraformaldehyde for more than 2 days and then embedded in paraffin. Serial sections of 4 μm thickness were cut and stained with an H&E staining kit (Nanjing Jiangcheng Bio Inc., Nanjing, China) and a Masson staining kit (Nanjing Jiangcheng Bio Inc., Nanjing, China). The quantification of the areas of fibrosis was measured by ImageJ software. Immunohistochemistry (IHC) was incubated with indicated first antibodies: anti-Bhlhe40 (1:100; 17895-1-AP), anti-α-SMA (1:200; 67735-1-Ig), anti-NLRP3 (1:200; 19771-1-ap) (Proteintech, Wuhan, HB, China), or anti-F4/80 (1:100; ab6640, Abcam, Cambridge, MA, USA) overnight at 4°C and then incubated with appropriate secondary antibodies for 30 mins at 37°C and detected using a DAB Kit (E-IR-R217, Elabscience Biotechnology Co., Ltd, Wuhan, China). Quantitative analysis of histologic staining was performed by ImageJ, as previously described (17 (link)).