Figure 4. M18BP1 and CENP-C interact in vitro. (A) Co-localization of RFP-CENP-C and M18BP1-EGFP. K1B2 cells were transfected with a RFP-CENP-C expression construct. Maximum intensity projections of two representative staining patterns are shown. Scale bar is 5µm. (B) Co-immunoprecipitation of CENP-C and M18BP1. HEK293FT cells were transfected with expression plasmids for EGFP-CENP-C and myc-M18BP1. Nuclear extracts from these cells were incubated with agarose beads (control) and GFP-Trap affinity beads to enrich for EGFP-CENP-C and interacting bound proteins. Protein gel blot analysis shows the nuclear extract (Inp), proteins bound to agarose beads (mock) and proteins that were enriched with GFP-Trap agarose beads (IP). An empty lane is indicated by “-”. EGFP-CENP-C and myc-M18BP1 were detected using antibodies against GFP and myc, respectively. (C) Interaction tests between M18BP1 and CENP-C truncations. The scheme shows the domain structure of mouse CENP-C and the truncation constructs that were used in this assay. Recombinant GST-tagged M18BP1 truncations (M1-M5) were incubated with in vitro translated myc-CENP-C truncation proteins and bound to GST beads. The bound CENP-C protein truncations were detected using myc antibody. Only the C-terminal CENP-C fragment showed clear interaction with M18BP1. The M18BP1 fragments M1-M5 are depicted in Figure 3A.