CEP295 acts downstream of CPAP. (A) Schematic of the protocol used to analyze the recruitment of centriolar proteins in siRNA-treated PLK4–myc-inducible cells at early S stage. (B) Immunoblot analysis of cell lysates from siRNA-treated cells. (C) Depletion of CEP295 (i-v) did not affect the localizations of CPAP (ii), CEP120 (iii), centrobin (iv) or SPICE (v) to new-born centrioles (NBC). PLK4–myc-inducible cells were treated with sicontrol or siCEP295 as described in A, analyzed by immunofluorescence microscopy, and the results quantified. (D) Depletion of CPAP interferes with the targeting of CEP295 to new-born centrioles (NBC). PLK4–myc-inducible cells were treated with siRNAs against CPAP, CEP120, centrobin or SPICE as described in (A) and analyzed by immunofluorescence microscopy. Scale bars: 0.5 μm. Error bars in C,D represent the mean±s.e.m. n=3 independent experiments each scoring 100 cells.