The C-terminal CM2-containing region of CDK5RAP2 is required for PCM foci formation and acentriolar mitosis. (A) Schematic of CDK5RAP2 highlighting predicted coiled-coils and the conserved CM1 and CM2 regions. The ΔC mutant deletes the C-terminal 168 amino acids that contain CM2. (B) Protocol for combining centrinone treatment and replacement of endogenous CDK5RAP2 with RNAi-resistant engineered variants. (C) Immunoblotting of the indicated conditions; mClover was detected using an anti-GFP antibody. α-Tubulin is a loading control. (D) Summary of mitotic outcomes in live imaging of the indicated conditions. (E) Mitotic duration for the indicated conditions. Error bars are 95% CI. P values are from t tests. (F) Immunofluorescence of acentrosomal CEP192 foci formation in fixed mitotic cells lacking centrioles. A centrin antibody was used to confirm centriole depletion. Scale bar, 10 µm. ***, P < 0.001; ****, P < 0.0001. IB, immunoblot; IF, immunofluorescence; ns, not significant; tub, tubulin.