Sds22 functions at kinetochores. (A) Intracellular localization of Sds22-GFP. Immunofluorescence of HeLa cells fixed 48 h after transient transfection with GFP-Sds22 plasmid. Overlay shows DAPI or anti–human centromere antibody (ACA; blue), Sds22-GFP (green), and microtubules (red). (B) Stable expression of Sds22-GFP in HeLa cells. Western blot analysis of extracts from untransfected HeLa cells and cell clone D103 stably expressing GFP-Sds22 probed with polyclonal Sds22-specific antibody. (C) Relative localization of Sds22 and PP1 at the kinetochore. Immunofluorescence of HeLa cells transiently transfected with mRFP–PP1-γ and GFP-Sds22 fixed 48 h after transient transfection. Overlay shows mRFP1–PP1-γ (red), Sds22-GFP (green), and aurora B (blue). (D) Relative localization of Sds22 and PP1 at the kinetochore. Immunofluorescence of HeLa cells transiently transfected with GFP-Sds22 plasmid fixed 48 h after transient transfection. Overlay shows Sds22-GFP (green), ACA (blue), and anti-tubulin (red). (E) Dependency of PP1 kinetochore localization on Sds22. Kinetochore localization GFP–PP1-β and GFP–PP1-γ were scored in mitotic spreads of HeLa cells 48 h after transfection with Sds22-specific and control siRNA. Data are from three separate experiments testing localization of either GFP–PP1-β or GFP–PP1-γ. Total number of cells for GFP–PP1-β: control, 47; Sds22 RNAi, 61. Total number of cells for GFP–PP1-γ: control, 162; Sds22 RNAi, 134. P < 0.001. P-values were calculated by Fisher’s exact test. (F) Dependency of Sds22-GFP kinetochore localization on PP1 isoforms. Relative number of cells showing EGFP-Sds22 at kinetochores in mitotic spreads of a stable HeLa cell line (clone D103) 48 h after transfection with control or PP1 isoform–specific RNAi. Data are from five independent experiments. Total number of cells and p-values: control, 93; PP1-α, 40 (P < 0.001); PP1-β, 84 (P = 0.024); PP1-γ, 81 (P = 0.001). Error bars indicate SEM. Bars: (A, top row) 10 µm; (A, rows 2–6) 5 µm; (C and D) 2 µm.