Figure 5. Immunoseparation of caveolae from DRM and differential localization of SNAP-23 and C8ORF2.

Caveolae were immunoseparated from isolated DRM (∼25 μg) in triplicate using CAV1 mAb (clone 2234) cross-linked to magnetic beads. Bound (B) and unbound (UB) fractions were resolved by SDS/PAGE and immunoblotted for CAV1, SNAP-23 and C8ORF2. (A) Using anti-CAV1 clone 2234, approx. 75% of total CAV1 can be captured, while CAV1 remains in the unbound fraction when using either an irrelevant endothelial-specific antibody (vWF) or no antibody. Most of the SNAP-23 remains in the unbound fraction, while C8ORF2 is co-immunoisolated with CAV1. (B) Quantification of the reproducible CAV1 immunoisolation efficiency and divergent capture ratios of SNAP-23 and C8ORF2, compared with CAV1.