Fig. 1.
Light-Induced Recruitment of INAD-Signaling Complexes to Lipid Rafts. Top and Middle, Membranes from either dark-raised or light-exposed wild-type flies were treated with 1% Triton X-100 at 4°C, followed by floatation through Optiprep density gradients (see Experimental Methods). Shown are representative immunoblots of the six fractions collected from top (1) to bottom (6) of density gradients. In dark-raised flies (Dark), all phototransduction components examined were found primarily in heavier density fractions #4–6. With light-exposure (Light; see Experimental Methods), INAD, and its target proteins, PLC, TRP, and eye-PKC, were recruited to detergent-resistant light density fractions (#1–2). In contrast, Rh1 and Gqα from membranes of both dark-raised and light-exposed flies were solubilized and found primarily in fractions #5–6. Bottom, Membranes from light-exposed wild-type flies were treated with 1% Triton X-100 at 37ºC and assayed in similar Optiprep density floatation gradients. Shown is a representative immunoblot of density gradient fractions. Recruitment of signaling components to light density fractions is almost completely abolished with detergent treatment at 37ºC, consistent with the proposal that INAD-signaling complex components are indeed translocating to DRM raft domains.