Dynein/Dynactin Pulldown Assay with Rab45 and CRACR2a

Pulldown of dynein/dynactin was performed as previously described (Huynh and Vale, 2017 (link)). Briefly, dynein/dynactin isolated from RPE1 cells were mixed with 20 nM purified Rab45 or CRACR2a and 15 µl preequilibrated Streptactin Sepharose beads (GE Healthcare) in 300 µl binding buffer (50 mM Hepes, pH 7.4, 20 mM NaCl, 1 mM Mg-Acetate, 10% glycerol, 0.1% NP-40, and 2 mM DTT). The mixture was incubated at 4°C for 1 h. Beads were pelleted at 1,000× relative centrifugal force for 2 min and washed four times with 500 µl binding buffer. The proteins were eluted by boiling in SDS loading buffer for 5 min and loaded onto SDS-PAGE gel. The amounts of CRACR2a and Rab45 in the pulldown samples were visualized by Coomassie staining. The amounts of dynein and dynactin were detected via Western blot with antibodies against dynein heavy chain and the p150 subunit of dynactin, respectively. In the pulldown assay shown in Fig. 2 A, 2 mM EGTA or EGTA:Ca²⁺ was added to the binding buffer to deplete calcium or to maintain a free calcium concentration of 2 µM. Information about antibodies used can be found in Table S1.