Connexin 43 (CX43), encoded by the GJA1 gene, is the most ubiquitously expressed member of the connexin family, a class of transmembrane proteins critical for gap junction intercellular communication (GJIC). Primarily localized at the plasma membrane, CX43 assembles into hexameric hemichannels; docking of hemichannels between adjacent cells forms gap junction channels, which permit the passage of small molecules (e.g., ions, metabolites, second messengers) and facilitate synchronized cellular activities. Beyond GJIC, CX43 hemichannels also function independently to mediate paracrine signaling by releasing signaling molecules (e.g., ATP) into the extracellular space. CX43 is widely distributed in cardiac tissue, smooth muscle, epithelial cells, and the central nervous system, and its dysregulation is linked to various pathological conditions, including cardiac arrhythmias, cancer metastasis, and neurodegenerative disorders. Moreover, CX43 physically interacts with Caveolin-1 (CAV1) at the plasma membrane, which modulates CX43 trafficking, channel gating, and degradation to fine-tune intercellular communication efficiency. Briefly,rat CAV1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CX43-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CAV1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. Measured by its binding ability in a functional ELISA. When Recombinant mouse CX43 is lmmobilized at 2 µg/mL(100 uLwell), the concentration of rat CAV1 that produces 50% optimal bindingresponse is found to be approximately 0.0015 µg/mL.