-tubulin was used as a loading control. DAPI (blue) and antibodies against Flag (red) and -tubulin (green). Control denotes expression of an irrelevant Flag-tagged protein. NIHMS6343-supplement-3.pdf (7.0M) GUID:?605D16DC-EE94-4C21-910F-42915DB736A0 4: Figure S4 A ~1.5 fold overexpression of Flag-Cep76 is sufficient to rescue centriole amplification induced by loss of Cep76 or HU treatment. (A) U2OS cells were transfected with control siRNA (siNSp) or siRNA targeting Cep76 3UTR and the indicated amounts of plasmid expressing Flag-Cep76. (Top) Western blotting of endogenous and recombinant Cep76 using an anti-Cep76 antibody. -tubulin Nedisertib was used as a loading control. (Bottom) The percentages of transfected cells with 4 CP110 dots were decided. (B) U2OS cells expressing increasing amounts of Flag-Cep76 were either left untreated or treated with HU. (Top) Western blotting of endogenous and recombinant Cep76 using an anti-Cep76 antibody. -tubulin was used as a loading control. (Bottom) The percentages of transfected cells with 4 CP110 dots were decided. In (A and B), at least 75 transfected cells were scored per condition, and common of three impartial experiments is shown. Error bars represent standard errors. Densitometric analyses were performed with ImageJ. NIHMS6343-supplement-4.pdf (438K) GUID:?026D847D-40AE-4324-A0B5-EDD543C922B2 5: Figure S5 A cancer-associated Cep76 S83C mutation is unable to suppress centriole amplification. U2OS cells transfected with control siRNA (siNSp) or siRNA targeting Cep76 3UTR (siCep76) and plasmid expressing an irrelevant Flag-tagged protein (control), Flag-Cep76 wild type (Cep76 FL) or mutant (Cep76 S83C). (A) The percentages of transfected cells with 4 CP110 dots were decided. At least 75 transfected cells were scored per condition, and average of three impartial experiments is shown. Error bars represent standard errors. (B) Cells were stained with DAPI (blue) and antibodies against Flag (red) and CP110 (green). NIHMS6343-supplement-5.pdf (1.8M) GUID:?0769A7FC-F5C1-4208-A292-6AB93952DE15 6: Figure S6 Cep76 slightly but not dramatically affects CDK2 and Cyclin A localization to the centrosome. (A) Fluorescence intensity of endogenous CDK2 at the centrosome was measured in U2OS cells transfected with control siRNA (siNSp) or Cep76 siRNA. (B) Fluorescence intensity of endogenous cyclin A at the centrosome was measured in U2OS cells transfected with control siRNA (siNSp) or Cep76 siRNA. In (A and B), at least 75 transfected cells were scored per condition, and common of three impartial experiments is shown. Error bars represent standard errors. NIHMS6343-supplement-6.pdf (342K) GUID:?4D8286E2-D20B-40E9-B535-7DA04B5964B4 7: Physique S7 Cep76 antagonizes Plk1 function to suppress centriole amplification. (A) (Left) U2OS cells were transfected with control siRNA (siNSp) or Cep76 siRNA and either left untreated or treated with BI 2536. The percentages of transfected cells with 4 centrin dots were determined. (Right) Cells were stained with DAPI (blue) and antibodies against Cep76 (red) and centrin (green). (B) U2OS cells transfected with plasmids expressing Flag and Myc, Flag-Cep76 and Myc, or Flag-Cep76 and constitutively active Plk1 (Myc-Plk1-T210D), were either left untreated or treated with HU. The percentages of transfected cells with 4 CP110 dots were decided. (ACB) At least 75 transfected cells were scored per condition, and average of three impartial experiments is shown. Error bars represent standard errors. NIHMS6343-supplement-7.pdf (723K) GUID:?C71EBC77-78C8-41AB-BECF-176B4D4D79A2 8: Figure S8 Flag-Cep76 is usually acetylated binding experiments using purified proteins revealed that while Cep76 associates weakly with cyclin E/CDK2, it binds strongly to cyclin A/CDK2 (Figure 1E), in close Nedisertib agreement with our immunoprecipitation results (Figure 1B). Taken together, these findings suggest that Cep76 preferentially interacts with cyclin A/CDK2. Open in a separate Nedisertib window Physique 1 Cep76 interacts with cyclin A/CDK2. (A) HA (control) or CDK2-HA was expressed in HEK293 cells and immunoprecipitated from lysate with an Rabbit polyclonal to LDLRAD3 anti-HA antibody. The resulting immunoprecipitates were Western blotted with anti-HA or anti-Cep76 antibodies. IN, input. (B) Flag (control) or Nedisertib Flag-Cep76 were expressed in HEK293 cells and immunoprecipitated with anti-Flag beads. Immunoprecipitates were Western blotted with the indicated antibodies. IN, input. (C) Flag or Flag-Cep76 and myc or myc-cyclin A were co-expressed in U2OS cells and Flag proteins were immunoprecipitated. Flag-Cep76 and myc-cyclin A were detected after western blotting the resulting immunoprecipitates. IN, input. (D) Western blotting of endogenous Cep76, CDK2 and Cyclin A after immunoprecipitation of U2OS cell extracts.