Journal: The Journal of Biological Chemistry

Article Title: Role of the endothelial caveolae microdomain in shear stress–mediated coronary vasorelaxation

doi: 10.1074/jbc.M117.786152

Figure Lengend Snippet: Subcellular distribution of SK, IK, and TRP channels in freshly isolated nonpassaged BCECs is shown. A, the subcellular distribution of SK1, SK2, SK3, IK, TRPC4, and TRPV4 in freshly isolated BCECs was measured by density gradient fractionation, with fraction 1 being the lightest and fraction 12 the heaviest. Western blots against anti-SK1, anti-SK2, anti-SK3, anti-IK, anti-TRPV4, anti-TRPC4, and anti-Cav-1 antibodies show that SK1, SK3, and TRPV4 are detected in the low-buoyant density, caveolae-rich fractions, whereas SK2, IK, and TRPC4 are mainly found in the high-density non-lipid raft fractions and absent in the caveolae-rich fractions. B, immunoprecipitates of anti-Cav-1 antibody after incubation with the low-buoyant density fraction 5 were blotted against anti-SK1, anti-SK3, anti-IK, and anti-TRPV4 antibodies. Pulldowns using nonimmune IgG served as controls. C, immunoprecipitates of anti-TRPV4 or anti-SK3 antibodies from the low-buoyant density fraction 5 were blotted against anti-Cav-1 antibodies. Pulldown using nonimmune IgG served as controls. D, BCEC in situ proximity ligation assay (PLA). The fluorescent signal generated by labeled complementary oligonucleotide probes after a 100-min amplification reaction at 37 °C in BCECs treated with mouse anti-Cav-1 and rabbit anti-SK3 or rabbit anti-TRPV4 antibodies. The nuclei were counterstained with DAPI, and the PLA signals were visualized at 40× magnification under a Zeiss 510 Meta Confocal Laser Scanning Microscope equipped with DAPI/Texas Red filters and analyzed using Zeiss 510 software.

Article Snippet: For immunoblotting, the proteins were boiled with 15 μl SDS-PAGE loading buffer at 100 °C, resolved by polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and then blotted against rabbit anti-Cav-1, rabbit anti-SK1 (1:200, Alomone Labs, APC-039), rabbit anti-SK2 (1:200, Alomone Labs, APC-028), rabbit anti-SK3 (1:200, Alomone Labs, APC-025), anti-TRPC4 (1:200, Alomone Labs, ACC-018), rabbit anti-TRPV4 (1:200, Alomone Labs, ACC-034), and anti-TRPC4 (Alomone Labs, ACC-018) antibodies.

Techniques: Isolation, Fractionation, Western Blot, Incubation, In Situ, Proximity Ligation Assay, Generated, Labeling, Amplification, Laser-Scanning Microscopy, Software