Figure 9.

The cytosolic p22–microtubule-binding factor behaves as 70- to 30-kDa globular protein upon fractionation on Superose 12. (A) Cytosol was fractionated on Superose 12 (fractionation range: 1–300 kDa) and pooled, or single fractions were analyzed by SDS-PAGE and immunoblotting with anti-p22. p22 elutes in fractions 31–33. (B) Pooled or single fractions were tested for their ability to support the binding of p22-myr to microtubules in the cosedimentation assay. Fractionation of the cytosol yielded a peak of p22–microtubule-binding activity around 70–30 kDa (fractions 29–31). Coomassie blue-stained SDS-PAGEs were scanned, and the amount of p22-myr found associated with the microtubule pellet was quantitated using the NIH Image program. (C) Immunoblotting of microtubule pellets with anti-CLIP-170 (α55), anti-p58, and anti-Tau (Tau 1) shows that the microtubule-binding profiles of these proteins do not overlap with the p22–microtubule-binding activity.