Ab82477

Total proteins were extracted from placental tissues using a protein extraction kit (Beyotime, Beijing, China) as informed by the manufacturer. Briefly, 10 μg of protein was loaded and separated by SDS-PAGE gel electrophoresis, and then the protein was transferred onto a polyvinylidene difluoride membrane (Merck Millipore). After blocking with TBST buffer containing 5% milk, the blots were then incubated overnight at 4 °C with each of the following primary antibodies: angiogenin (Ang) (ab95389, abcam, 1:1000), ADORA1 (ab82477, abcam, 1:1000), ADORA2A (ab3461, abcam, 1:1000), ADORA2B (ab222901, abcam, 1:1000), ADORA3 (ab197350, abcam, 1:1000), vascular endothelial growth factor A (VEGF-A) (19003-1-AP, Proteintech, USA, 1:1000), Akt (9272, CST, 1:1000), p-Akt (4060, CST, 1:1000), signal transducer and activator of transcription-3 (Stat3) (ab76315, Abcam, USA, 1:1500), p-Stat3 (ab68153, Abcam, USA, 1:1500), vascular cell adhesion molecule-1 (VCAM1) (ab134047, abcam, 1:1000), and β-actin (4970, CST, USA, 1:1000). Subsequently, the membranes were incubated with appropriate HRP-conjugated anti-rabbit IgG secondary antibody (AS014, Abclonal, China, 1:5000). Images were captured using the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA), and band densities were quantified using Image Lab soft-ware (Bio-Rad, Hercules, CA, USA) and then normalized to β-actin content.

For immunofluorescence examination, the cardiomyocytes or myocardial tissues were fixed with paraformaldehyde, followed by treatment with 0.5% Triton X-100. After blocking with 5% BSA at room temperature for 1 h, the samples were incubated overnight at 4 °C with primary antibodies against α-actinin (1:250), A1AR (1:250), A2aAR (1:250) and CTNT (1:250), followed by incubation with Alexa Fluor 488 (1:500), Alexa Fluor 555 (1:500), Alexa Fluor 594 (1:500) or Alexa Fluor 647 (1:500) secondary antibodies at room temperature for 90 min in the dark. Nuclei were counterstained with DAPI (1:1000). After labeling and washing, fluorescent photographs were acquired through a Zeiss LSM 880 confocal microscope (Carl Zeiss, D07740 Jena, Germany) under 488 nm, 555 nm, 594 nm and 647 nm excitation. Fluorescence intensity and cell surface area were quantified using the ZEN imaging software (Carl Zeiss, D07740 Jena, Germany). The following primary antibodies were used in this study: A1AR (ab82477; Abcam, Cambridge, UK), A2aAR (ab79714; Abcam), and α-actinin (ab9465; Abcam); CTNT(15513-1-AP, Proteintech, Chicago, IL); DAPI (C1006, Beyotime, Shanghai, China); Alexa Fluor 488 (AS053, ABclonal, Wuhan, China); Alexa Fluor 555 (AS057, ABclonal, Wuhan, China); Alexa Fluor 594 (AS054, ABclonal, Wuhan, China); Alexa Fluor 647 (AS059, ABclonal, Wuhan, China).

Extracts of proteins from cells and tissues were prepared using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined by BCA protein assay kit (Beyotime, Shanghai, China). About 25 µg of total protein was separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with 5% BSA for 1 h, the membranes were incubated with primary antibodies against A1AR (1:1000), A2aAR (1:500) and GAPDH (1:8000) overnight at 4 °C, followed by incubation with secondary antibody (1:2000) or (1:10,000) for 1 h. The blots were visualized via chemiluminescent detection (Immobilon Western HRP, Millipore, Billerica, MA), and analysed using Alpha View SA software (Alfaview Gmbh, Karlsruhe, Germany). The following primary antibodies were used in this study: A1AR (ab82477; Abcam, Cambridge, UK), A2aAR (ab79714; Abcam) and GAPDH (60004-1-Ig; Proteintech, Chicago, IL).