Figure 6.

T-synthase from cells in the absence of a functional Cosmc is mainly in covalent oligomers. T-synthase–HPC4 expressed in LSC and LSB cells treated with 10 μM lactacystin was affinity purified and analyzed on SDS-PAGE under reducing and nonreducing conditions and Western blotted with anti-HPC4 (A). Cell extracts were made and cross-linking was performed by adding 5 mM DSS. The recombinant T-synthase was purified and analyzed by Western blotting under reducing conditions (B). Sf-9 cells infected with Baculovirus-expressing T-synthase-HPC4 or coinfected with Baculovirus encoding wtCosmc were harvested and one portion was made for cell extracts. The cell extracts and the other portion of intact cells were treated with DSS. T-synthase was purified from the cell extracts and analyzed by Western blot with anti-HPC4 (C). Black line indicates that intervening lanes have been spliced out.