Figure 5.

Dimerization of ALG-2. In panel A, The cell-permeable cross-linker DSP was added to Mel290 cells pretreated with thapsigargin. Cells were lysed and analyzed by immunoblotting using anti-ALG-2 antibodies. Lane 1 contains cross-linked cell lysate; lane 2 contains cross-linked cell lysate solubilized in the presence of β-mercaptoethanol. In panel B, the absorbance at 280 nm was recorded for three different concentrations of ALG-2 during centrifugation in the presence of 1 mM EDTA. The natural log of the absorbance values at equilibrium were plotted against the radial position in the three cells during centrifugation at 9000, 12 000, 16 000, and 22 000 rpm. In panel C, a similar plot was generated from data obtained for dNALG-2 (5 μM Ca²⁺). Bold lines show the overlay of recovery values measured at 12 000 rpm after centrifugation at higher speeds with initial 12 000 rpm values, indicating little loss in protein due to irreversible precipitation. The sedimentation equilibrium curves were fitted assuming the presence of a single thermodynamically ideal species. Data were fitted as outlined in the Materials and Methods. Cross-linking data represent one of four independent experiments, while analytical centrifugation data were gathered from duplicate experiments.