Figure 2

Analysis of detergent-free isolated lipid rafts by raft ELISA and SDS-PAGE. Anti-Thy-1, which recognizes a GPI-anchored protein known to be a raft constituent, Thy-1, was used as the capture antibody that coated the microtitre wells. Wells were incubated with the detergent-free fractions isolated from YH16.33 cells pretreated (Tx) with MβCD (A), or left untreated (No Tx) (A & B). Captured rafts were detected by biotinylated anti-Ly-6 antibody (A) or anti-TCRαβ antibody (B), followed by avidin-HRP. The assay was developed by adding ABTS peroxidase substrate and the absorbance was read by a Spectramax 190 plate reader. Fractions were also analyzed by running SDS-PAGE on 4-20% Tris-HCl gels followed by the transfer of proteins onto a PVDF membrane. Membranes were probed with anti-LAT or anti-β-COP (C), followed by the appropriate secondary antibodies. GM1 was detected in these fractions by dot-blot on PVDF membrane with biotin-cholera toxin β and avidin-HRP conjugate (D). Blots were observed using a chemiluminescence kit (Pierce, Rockford, IL) as described in the Materials and Methods section. Shown is a representation of at least three independent experiments.