(A) The CENP-M alignment identifies highly conserved residues (based on alignment in Figure 3—figure supplement 1, panel D), a subset of which (asterisks) is exposed at the surface of CENP-M. (B) Position of conserved residues shown in A on two opposite faces of the CENP-M surface. (C) After insect cell co-expression of indicated proteins, affinity purification with GST-CENP-M (if present) led to isolation of associated proteins shown, after SDS-PAGE separation, in the ‘beads’ fraction. Material eluted from beads was collected and shown in lanes labeled ‘elution’. Co-expression of all four CENP-HI57–CKM subunits (positive control) is necessary for identification of the CENP-HI57–CKM complex on beads and in elution fraction. CENP-ML94A–L163E fails to assemble CENP-HI57–CKM despite the expression of CENP-H, CENP-I57–C and CENP-K. (D) GFP-CENP-Mwt, but not GFP-CENP-ML94A+L163E, co-immunoprecipitates CENP-I, CENP-T and Mis12 from mitotic cells. Panels represent the α-GFP co-immunoprecipitation analysis of protein extracts obtained from mitotic HeLa Flp-In T-REx cells stably expressing GFP, GFP-CENP-Mwt or GFP-CENP-ML94A+L163E from an inducible promoter. Total protein extracts (Input) and immunoprecipitates (α-GFP IP) were run on SDS-PAGE and subjected to WB with indicated antibodies. Vinculin was used as a loading control. (E) Representative images of HeLa Flp-In T-REx cells treated with siRNA for endogenous CENP-M and expressing the indicated siRNA-resistant GFP-CENP-M fusions. Expression of GFP-CENP-Mwt, but not of GFP-CENP-ML94A+L163E, rescues chromosome alignment defects and loss of CENP-I kinetochore localization observed upon depletion of endogenous CENP-M. Following fixation, cells were immunostained and imaged for GFP, CENP-I, CREST and DNA (DAPI). Insets show a higher magnification of regions outlined by white boxes. Scale bars = 2 µm. (F) Quantification, for experiment in E, of the CENP-I kinetochore levels normalized to CREST kinetochore signal. Graphs and bars indicate mean ± SEM. See ‘Materials and methods’ section for details on quantification.