Figure 1: A centrosomal protein-interaction network identifies new centrosomal proteins.

(a) Protein–protein interaction subnetwork derived from the complete TAP interaction network (Supplementary Data 1) that displays known, newly identified or newly localized centrosome proteins (LGALS3BP and MAGED2). Interactions are represented as edges between bait proteins and their interaction partners. The bait proteins are displayed as hexagons, centrosomal proteins are coloured in grey and centriolar proteins in red. High-confidence interactions (Mascot score of the prey protein >50, at least two identified peptides) are shown with solid lines, and candidate interactions (Mascot score >24) are shown with dotted lines. (b,c) The protein–protein interactions of the main targets were validated by either reverse TAP (b) or co-immunoprecipitation (c). (b) The interactions of NME7 with TUBG1, TUBGCP2 and TUBGCP3 were confirmed by reverse TAP. The bait protein NME7 is identified through its TAP-tag moiety calmodulin-binding site (CBS). The cell extract was used as a positive western blotting control for the identification of the three NME7-interacting proteins. (c) The interaction between LGALS3BP and TUBGCP3 was confirmed by co-immunoprecipitation after co-expression of FLAG-TUBGCP3 and Myc-LGALS3BP in HEK293 cells. (d) The interactions between Myc-tagged LGALS3BP and a Flag-tagged CEP250 fragment co-expressed in HEK293 cells were confirmed by Flag-immunoprecipitation. Flag-tagged EGFP co-expressed with Myc-tagged LGALS3BP were used as negative control of the Flag-IP. (e) Endogenous MAGED2 interacts with TAP-tagged LGALS3BP. TAP-tagged EGFP was used as a negative control in the TAP experiments. (f) Examples of Coomassie stained 1-cm long SDS–PAGE gels of TAP eluates of centrosome-related bait proteins. The samples show the complexity of centrosomal protein-interacting proteins subjected to MS identification, in comparison with control samples (mock purification).