Figure 7.

Analysis of STxB association with DRMs along the retrograde pathway. (A–C) Cy3-labeled STxB was internalized for 45 min at 19.5°C (A) or 37°C (B), or Cy3-STxB-Glyc-KDEL (C) for 4 h at 37°C into HeLa cells. The cells were then either fixed directly (−Triton X-100), or preextracted with 1% Triton X-100 solution (+Triton X-100) before fixation. Permeabilized cells were then stained for GM₁ and the TfR. Under these conditions, GM₁ and the EE- (A), Golgi- (B), or ER (C)-associated STxB resisted extraction, whereas the TfR was readily extracted. (D–E) STxB-Sulf₂ was internalized in the presence of radioactive sulfate for 45 min at 37°C into HeLa cells (D), and iodinated STxB-Glyc-KDEL was internalized for 15 h (E). The cells were then washed, lysed in 1% Triton X-100 buffer, and DRMs were floated in Optiprep step gradients. The gradients were fractionated, STxB in the fractions was immunoprecipitated with 13C4 antibody, and immunoprecipitates were analyzed by gel electrophoresis. DRM fraction 2 contained a significant amount of the sulfated (D) or glycosylated (E; uppermost bands) STxB.