Subcellular Fractionation and Immunoblotting

The cells grown to confluence in 15-cm dishes were washed with PBS, scraped, pelleted, and centrifuged at ~800 x g for 1 min at 4°C. After discarding the supernatant, SPE Buffer I from the FOCUS™ SubCell kit (G-Biosciences, St Louis, MO, USA) was added to the pellet. The samples were vortexed and incubated on ice for 10 min. Then, the cells were lysed using a Dounce homogenizer with 20 strokes per sample. To pellet the nuclei, the samples were centrifuged at 700 x g for 10 min at 4°C. The supernatant was transferred to a new tube and subsequently centrifuged at 12,000 x g for 15 min at 4°C to pellet the mitochondria. The remaining supernatant was transferred to a new tube and subsequently centrifuged at 100,000 x g for 60 min at 4°C in a SW50.1 swinging bucket rotor (Beckman Coulter™, Palo Alto, CA, USA), to pellet the membrane fraction. The plasma membrane-enriched pellet was subjected to detergent-free sucrose gradient ultracentrifugation (16 (link),23 (link)–26 (link)). Twelve fractions were collected and immunoblotted for N-Myc-tagged D₁R and CAV-1.