Before cross-linking, the dynein fractions from sucrose density gradients were dialyzed against J-buffer containing 200 mM potassium acetate overnight at 4°C. BS3 cross-linker (Thermo Fisher Scientific) was added to aliquots of the dialyzed dynein fractions from sucrose density gradients and incubated for 30 min at 25°C. The cross-linking reactions were terminated by addition of 20 mM glycine (pH 7.8). The mixtures were dialyzed against a UV buffer [450 mM sodium acetate, 0.5 mM EDTA, 1 mM dithiothreitol, 2 mM MgCl2, 10 mM Hepes (pH 7.4)] for 5 hours at 4°C. To perform vanadate-mediated photocleavage of the dynein HCs (52), 100 μM sodium metavanadate and 1 mM ATP were added to the dialyzed samples. The samples were placed in a 24-well plate on ice and irradiated with 365-nm light for 30 min. For SDS-PAGE, 4% polyacrylamide/4 M urea gels were used as the separating gel. The cross-linked products were detected by Western blotting using anti-DYBLUP antibody.

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