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. 2023 Sep 13;155(11):e202213217. doi: 10.1085/jgp.202213217

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© 2023 Akin et al.

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Figure 8. — ANO1, Ca_V1.2, and IP₃R colocalize in peripheral coupling sites to form signaling complexes. (A and B) Co-IP of Ca_V1.2 or IP₃R with ANO1 from lysates of the pulmonary artery from wild-type mice. Pulldown was carried out with anti-ANO1 antibody and then probed by Western blot with anti-Ca_V1.2, anti-IP₃R, or anti-ANO1 antibodies. Five to six mouse tissues per experiment, each ran in triplicates. (C and D) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and Ca_V1.2 (C) or ANO1 and IP₃R (D). All three proteins were preferentially localized to the periphery of the cells. (D and F) Line profiles of the areas indicated by the white dashed lines in C and E. The fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and Ca_V1.2 show strong immunolabeling at the PM (D). (E) IP₃R shows some intracellular immunolabeling, with moderate peaks present at the periphery showing an enhancement of protein localization to peripheral coupling sites. Source data are available for this figure: SourceData F8.