MISP is an actin-associated protein. (A) Localization of MISP (gray; red in merge) in interphase and different indicated mitotic stages of HeLa cells was shown by costaining with MISP antibody, Atto 488 phalloidin (green), and Hoechst 33342 (blue). Bars, 10 µm. Insets showed higher magnification of the framed regions. Bars, 5 µm. (B) HeLa cells were incubated with 2 µM of the actin-depolymerizing agent cytochalasin D (Cyto. D) for 10 min and analyzed by immunofluorescence. MISP, gray; red in merge; Atto 488 phalloidin, green. Bars, 10 µm. Insets showed higher magnification of the framed regions. Bars, 5 µm. (C) Actin cosedimentation assay with polymerized nonmuscle β-actin and GST-MISP or GST control. P, pellet; S, supernatant.