The human Nesd counterpart localizes to the cleavage site and interacts with centralspindlin. (A) HeLa cells were transiently transfected with a construct expressing SHCBP1::GFP and, after 48 h, fixed and stained to detect GFP, tubulin (tub), and DNA (blue in the merged image). (B) HeLa cells were transiently transfected with a construct expressing SHCBP1::GFP and, after 48 h, fixed and stained to detect GFP, RacGAP1, and DNA (blue in the merged image). (C) Extracts from HeLa cells transiently expressing either GFP or SHCBP1::GFP were immunoprecipitated using GFP antibodies (GFP IP). The Western blot on the left (INPUT) shows the level of expression of the GFP proteins. The presence of the two centralspindlin components MKLP1 and RacGAP1 in the SHCBP1::GFP pull-downs was confirmed by Western blotting. The position of IgG bands is indicated. The numbers on the left indicate the sizes in kilodaltons of the molecular mass markers. (D) HeLa cells transiently expressing SHCBP1::GFP were transfected with either control scramble or MKLP1 siRNAs for 48 h and then fixed and stained to detect GFP, tubulin, and DNA (blue in the merged image). The percentage of cells without any accumulation of SHCBP1 at the cleavage site (55%) or very weak and diffuse SHCBP1::GFP staining along CS microtubules (45%) is indicated. Bars, 10 µm.