Nesprin-2 recruits dynein–dynactin–BicD2 complex and kinesin-1 via separate but spatially close motifs. (A) Schematic of Nesprin-2 Giant (Nesprin-2G) and the truncated Nesprin-2 SR fragments highlighting the kinesin-binding motif and the putative dynein-dynactin-binding motifs. (B and C) Immunoblotting (B) and quantification (C) of co-immunoprecipitation (co-IP) of HEK293 cell lysate transfected with indicated Halo-tagged Nesprin-2 truncates. n = 3 independent experiments. Ratio paired t test. (D) Schematic of Nesprin-2 C-terminal truncates and co-IP results of HEK293 cells transfected with indicated Halo-tagged Nesprin-2 truncates. (E and F) Quantification of co-IP of endogenous p150 and KHC (E) and the p150/KHC ratio (F). n = 4 independent experiments. Ratio paired t test. (G) Schematic indication of the mutation sites. In SR48-5A-AAVV-KASH, all three Spindly motifs and one CC1-box motif were mutated. In SR48-LEAA-KASH, the LEWD motif was mutated. (H and I) Immunoblotting (H) and quantification (I) of co-IP with Halo-tagged wild-type and mutant SR48-KASH in HEK293 cell lysate. n = 5 independent experiments. Ratio paired t test. (J and K) Immunoblotting (J) and quantification (K) of co-IP with Halo-tagged SR48-KASH in control (Ctrl) or BicD2 knockout (KO) HEK293 cell lysates. BicD2 KO1 and KO2 are two independently isolated KO cell lines. n = 3 independent experiments. Ratio paired t test was used to compare with Ctrl. IB, immunoblotting; IP: immunoprecipitation. Bars show mean ± SEM. Source data are available for this figure: SourceData F2.