FIGURE 7:

BICD2-N-dependent motility requires LIS1. (A) Streptavidin pull-down assays with Bio–GFP or Bio–GFP–BICD2-N were analyzed with the indicated antibodies. Two percent of the cell lysate used for the IP and 25% of the IP sample were loaded on gel. (B) Scheme of the regulated heterodimerization constructs used to attach BICD2-N to endosomes. (C, D) HeLa cells were transfected with different siRNAs; 2 d later, cells were cotransfected with HA–BICD2-N–FRB and FKBP2-GFP-VAMP2; after one additional day in culture, cells were treated with rapalog, fixed, and stained for transferrin receptor. (C) Percentage of HA–BICD2-N–FRB– and FKBP2-GFP-VAMP2–coexpressing HeLa cells with endosomes fully clustered in the cell center, 30 min after rapalog addition. Approximately100 cells were analyzed in three independent experiments. (D) Representative images of HA–BICD2-N–FRB– and FKBP2-GFP-VAMP2–coexpressing cells in different conditions.