FIGURE 2:

Direct protein–protein interaction of nebulette and full-length desmin. A specific biochemical interaction of nebulette to desmin was detected under buffer conditions that restrict desmin filament maturation by sucrose gradient sedimentation. Soluble desmin oligomers and nebulette M1–5 recombinant proteins were precleared from small aggregates by ultracentrifugation. Desmin alone (A) or nebulette alone (B) or in a 1:1 M ratio mixture (C) were incubated for 1 h at 37°C and placed on top of 10–70% sucrose gradients made in a buffer (5 mM Tris-HCl, 1 mM EDTA, and 0.1 mM EGTA, pH 8.4) that favors desmin oligomer formation. After a 5 h sedimentation at 42,000 rpm, fractions (1–10 and pellet) were collected and analyzed by 12% SDS–PAGE and Western blot. Arrows point to bands for desmin or nebulette M1–5, migrating at their expected molecular weights of 52 and 25 kDa, respectively.