Anti cbs

Confluent HUVECs were starved overnight and were cultured in normoxic or hypoxic conditions. Then, cells were lysed in a 1 × sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% w/v SDS). Bicinchoninic acid (BCA) reagent was purchased from Shen Neng Bo Cai Corp. (Shanghai, China) and the BCA method was used for protein concentration determination. Protein samples (50 μg) were subjected to electrophoresis in a 10% SDS-polyacrylamide gel, and then transferred to a polyvinyl difluoride membrane (Millipore-Upstate Biotechnology, Lake Placid, New York, USA). After blocking in 5% non-fat milk in Tris Buffered Saline with Tween-20 (TBST, 0.05% Tween-20) for 2 h at room temperature (RT), the membrane was incubated with primary antibodies at 4°C overnight. Anti-CBS and anti-β-actin were purchased from Proteintech Group (Chicago, IL, USA). Anti-CSE and anti-MPST were purchased from Santa Cruz Biotechnology (CA, USA). After washing the membranes using TBST and then incubating them with horseradish peroxidase-conjugated secondary antibodies for 2 h at RT, SuperSignal West Pico Chemiluminescent Substrate (Thermo-Pierce Biotechnology, Rockford, USA) was used for band detection. Band signals were then quantified using Smart viewer software.

IHC was performed to evaluate the expression of CSE, CBS and 3-MST. Cardiac tissue sections were deparaffinized and rehydrated through a graded series of ethanol. Antigen retrieval was carried out using appropriate methods. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide. Non-specific binding was blocked using serum from the species in which the secondary antibody was raised. The sections were then incubated overnight at 4 °C with anti-CSE (Proteintech, USA, 1:200), anti-CBS (Proteintech,1:200) and anti-3-MST (Abclonal, USA, 1:200) antibodies. After washing, the sections were incubated with the corresponding secondary antibody, conjugated with a suitable enzyme such as HRP. Visualization of the protein expression was achieved using a chromogenic substrate, typically DAB. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted with a coverslip. Microscopic examination was performed, and images were captured for observation of staining intensity.

Heart tissues were homogenized in lysis buffer, and protein content was measured by using the BCA reagent. Equivalent amounts of protein were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore-Upstate). Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4°C in primary antibodies, anti-CSE (1:1,000, Proteintech, United States), anti-CBS (1:1,000, Proteintech, United States), anti-3-MST (1:500, Abcam, United States), anti-TLR4 (1:500, Abcam, United States), anti-CHOP (C/EBP homologous protein) (1:1,000, Abcam, United States), anti-GRP78 (78-kDa glucose-regulated protein) (1:1,000, Abcam, United States), anti-caspase-12 (1:1,000, Abcam, United States), anti-total PERK (1:1,000, Santa Cruz, United States), anti-pPERK (1:1,000, Santa Cruz, United States), anti-ATF6 (1:1,000, Santa Cruz, United States), anti-IRE-1α (1:1,000, Abcam, United States), anti-pIRE-1α (1:1,000, Abcam, United States), and β-actin (1:1,000, Proteintech, United States). The membranes were rinsed in tris-buffered saline and Tween 20 three times for 5 min and incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h. Specific bands were developed by using a chemiluminescence detection system (Thermo, Scientific-Pierce). The band intensity was quantified by Image J software.