FIGURE 4.
Transfected TUG generates and binds p97 monomers. A, HEK293 cells were transfected with FLAG-tagged TUG and lysed using 0.5% Nonidet P-40. Postnuclear supernatants were analyzed by sedimentation on sucrose density gradients, and fractions were immunoblotted to detect p97. The positions of molecular weight standards are indicated. The experiment was repeated with similar results. B, proteins in fractions 7 and 10 from the gradients in A were separated using native PAGE and then immunoblotted as indicated. Recombinant His6-p97 was included in the first lane for comparison, and the position of molecular weight standards is indicated at the left. Western blots (WB) of p97 and TUG were done on the same membrane and were imaged at different wavelengths using a LI-COR Odyssey imaging system. The experiment was repeated with similar results.