Figure 3.

Association of lipid raft proteins with detergent resistant membranes. Normal PF cells or those expressing autoantigen I/6-eGFP or eGFP-IFT27 were treated with 1% Triton-X at either 4 °C or 37 °C and separated into either soluble supernatant (Sup) or insoluble (Pellet) fractions via centrifugation. Fractions were analyzed by western blotting to determine DRM association, with proteins found in the pellet at 4 °C but the supernatant at 37 °C being in DRMs. Most proteins were detected with specific antisera, except for autoantigen I/6-eGFP and eGFP-IFT27, which were detected using anti-GFP. The following controls were included: Calflagin Tb44 and CAP5.5 are known DRM-associated proteins, mtHsp70 is a detergent soluble mitochondrial matrix protein, paraflagellar rod 2 protein (PFR) is a detergent insoluble cytoskeletal protein, and BIP (GRP78) can be considered a “loading control” since it is found in all fractions.