Lane maker loading buffer

The expression of target proteins in left atrial tissues was determined by western blotting. Proteins were homogenized in radioimmunoprecipitation assay lysis buffer (Solarbio Institute of Biotechnology, Shanghai, China). Protein concentrations were determined using an Enhanced Bicinchoninic Acid Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Solubilized proteins were denatured in Lane Maker Loading Buffer (CWBio, Beijing, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% gels, and transferred to polyvinylidene difluoride filter membranes (Beyotime Institute of Biotechnology). Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline at room temperature. After 2 h, membranes were incubated with primary antibodies at 4°C overnight. The following day, membranes were washed and incubated with a secondary antibody (AP132P; Nachuan Biotech, Changchun, China) for 2 h at room temperature. Bands were visualized with the ECL Plus Western Blotting Detection System (ECL Western Blot Kit, RB-SRCHLGT; Ray Biotech, Atlanta, GA, USA). The densitometry of western blots was quantified using ImageJ software (USA National Institute of Health, Bethesda, MA, USA).

Proteins from left atrial tissues were analyzed by western blotting. The atrial tissues were homogenized in radioimmunoprecipitation assay lysis buffer (Solarbio Institute of Biotechnology, Shanghai, China) and protein concentrations were determined using the Bradford Protein Assay Kit (BMG LABTECH, Offenburg, Germany). Solubilized protein was denatured in Lane Maker Loading Buffer (Cwbio, Beijing, China), separated by SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% or 8% gels and transferred to polyvinylidene difluoride filter membranes (Beyotime Institute of Biotechnology, Shanghai, China). Membranes were blocked with 5% nonfat dry milk in phosphate buffered saline (PBS) at room temperature. After 2 h, the membranes were incubated with rabbit phospho-PPARγ (S112) polyclonal antibody (1:1000; Elabscience, Wuhan, China) or with a rabbit polyclonal antibody to β-actin (1:1000; ComWin Biotech, Beijing, China) at 4℃ overnight. Membranes were incubated with secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) for 2 h at room temperature. After extensive washing with phosphate buffered solution (PBST), bands were visualized with the ECL Plus western blotting detection system (ECL Western Blot Kit; CWBIO, Taizhou, China) and then quantified using Image J software (USA National Institute of Health, Bethesda, USA).