Figure 2

Cby exists as a homodimer. (A) Gel filtration analysis of Cby. His-Cby purified from stable HEK293T cells was run on a pre-calibrated Superdex 75 FPLC column in buffer containing 1 M NaCl. An aliquot of each fraction was resolved by SDS-PAGE and analyzed by Western blotting using anti-Cby antibody. The arrows indicate the elution positions of protein standards: aldolase, 158 kDa; bovine serum albumin, 67 kDa; ovalbumin, 45 kDa and cytochrome C, 12 kDa. Similar results were obtained using buffer containing 0.5 M NaCl (data not shown). (B, C) Cross-linking experiments. His-tagged Cby was purified from transiently transfected HEK293T cells using Ni-NTA beads, and subjected to cross-linking with glutaraldehyde for 5 or 20 min (B), or dimethyl suberimidate (DMS) for 30 min (C). The samples were then resolved by SDS-PAGE, followed by immunoblotting with anti-Cby antibody.