Figure 10.

Velocity gradient centrifugation to determine the size of the cross-linked complex. Ag8(8) and Ag8.653 cells (10 × 10⁶) were metabolically labeled and the postnuclear fraction was prepared as described previously. Cross-linking was performed by including 150 μg/ml DSP in the HEPES buffer containing 0.25 M sucrose. Triton X-100 (1%) was added to the cross-linked sample and then the lysates and high-molecular-weight markers were centrifuged through 20–40% glycerol gradients. Each gradient was then separated into 15 fractions and the complex was precipitated with protein A-Sepharose beads for the Ag8(8) complexes (A) or immunoprecipitated with a polyclonal anti-BiP antibody for the Ag8.653 complexes (B).