Once the rat mesenteric LMCs were isolated and washed, they were incubated with Kir6.1, Kir6.2, SUR1, or SUR2 primary antibodies (Supplemental Table 1) for 1 hour at room temperature in IB. These antibodies target subunit-specific protein sequences, a claim that we substantiated by searching epitope sequences of antibodies using BLAST (https://blast.ncbi.nlm.nih.gov). The specificities of anti-Kir6.1 and anti-Kir6.2 antibodies also have been validated in cells from Kir6.1−/− and Kir6.2−/− mice (Milovanova et al., 2006). Subsequently, the cells were washed and incubated with goat anti-rabbit IgG secondary antibody Alexa 647 (1:500) and smooth muscle–specific α-actin–FITC (1:100) for 30–45 minutes. Then cells were washed three times with 1 ml of IB and resuspended in 300 µl IB for flow cytometry analysis. The data were collected using an Accari C6 flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo-V10 software (FlowJo LLC, Ashland, OR) to provide information on the expression of distinct KATP channel subunit proteins in isolated LMCs.

To visually confirm that the antibody signal arose from single LMCs and not contaminating cell types, we also subjected isolated LMCs to imaging flow cytometry. The LMCs were incubated with primary antibodies (Supplemental Table 1) corresponding to Kir6.1 subunit (rabbit anti-rat, 1:50) and Kir6.2 subunit (guinea pig anti-rat, 1:50) for 1–1.5 hours at room temperature in IB. Subsequently, the LMCs were washed and incubated with two secondary antibodies, goat anti-rabbit IgG Cy3 (1:2000) and goat anti-guinea pig Alexa 647 (1:2000), together with smooth muscle–specific α -actin–FITC (1:500) antibody, respectively, for 45 minutes. Then the cells were washed and resuspended in 50 µl of PBS. Finally, the suspensions of LMCs were imaged using an ImageStreamX Mk II Imaging Flow Cytometer (Luminex Corporation, Austin, TX). Images were collected using IDEAS 6.2 software (Luminex Corporation).

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