• Six commercial AT1R antibodies were validated using AT1R knockout rat and mouse models, overexpressing cell lines, and multiple assays.
• Only one antibody (A14201) consistently detected AT1R at the expected molecular weight across all tested tissues, while others showed tissue-specific or non-specific bands.
• The study highlights the lack of specificity in many commercial GPCR antibodies and provides a reliable validation protocol for AT1R detection.
• These findings are crucial for accurate AT1R research in cardiovascular diseases, ensuring reproducibility and reliability of antibody-based assays.