Ecl reagent

Total proteins from primary chondrocytes were extracted with RIPA buffer containing protease and phosphatase inhibitors (R0010, Beijing Solarbio Science & Technology Co., Ltd.) on ice. The concentration of proteins was measured by bicinchoninic acid (BCA) protein assay (Beyotime Institute of Biotechnology, Inc.). Equal amounts of protein (50 μg) were separated by 10 or 12.5% SDS-PAGE (PG112/PG113, EpiZyme, inc.) and transferred onto 0.22 μM NC membranes (P-N66485, Beijing Solarbio Science & Technology Co., Ltd.). The membranes were blocked with 5% dried skimmed milk in Tris-buffered saline with 0.05% Tween-20 (TBS-T) for 1 h at room temperature with sustained shaking. The membrane was washed with TBS-T 3 times then probed with primary antibodies (1:1,000) by incubation at 4°C overnight. The following day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary anti-bodies (1:5,000) for 2 h at room temperature. Immunoreactivity was visualized using ECL reagent (Wanlei Biotechnology) using ChemiDoc™ MP Imaging System (Tanon Science & Technology Co., Ltd.). The densities of the bands were quantified using a Bio-Rad Chemi EQ densitometer and Quantity One software (Tanon Science & Technology Co., Ltd.).

Briefly, the total protein was collected from H9c2 cells and separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Subsequently, the proteins were electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and incubated overnight (4°C) with primary antibodies (Table 1). After that, the membranes were washed and incubated with appropriate secondary antibodies for 2 hours (37°C). Protein bands were developed using an enhanced chemiluminescence (ECL) reagent (Wanleibio, Shenyang, China), and the band intensity was quantified using Gel-Pro Analyzer Software (Media Cybernetics, Bethesda, MD, USA). β-actin served as an internal loading control. The relative protein level was determined as the ratio of the gray value of the target band to that of the β-actin band.
Table 1

Primary Antibodies Used in Western Blot

Antibody	Company	Catalog No.	Dilution
SIRT3	Santa Cruz	sc-365175	1:100
FoxO3a	Wanleibio	WL02891	1:1000
Cleaved caspase-3	Wanleibio	WL01992	1:500
Bax	Wanleibio	WL01637	1:500
Bcl-2	Wanleibio	WL01556	1:1000
Parkin	Wanleibio	WL02512	1:500
Beclin1	Wanleibio	WL02508	1:500
NIX	Affinit	DF8163	1:500
LC3 I/LC3 II	Wanleibio	WL01506	1:1000
p62	Wanleibio	WL02385	1:400
MT2	Abcam	ab203346	1:500
β-actin	Wanleibio	WL01372	1:500