Sequential phosphorylation of Nedd1 by Cdk1 and Plk1 is required for targeting of the TuRC to the centrosome

JCS042747 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Specificity of mouse and rabbit anti-Nedd1 antibodies determined by immunofluorescence microscopy. (A,B) Interphase and mitotic HeLa cells were double-labeled for γ-tubulin and Nedd1 (A, mouse anti-Nedd1; B, rabbit anti-Nedd1). Scale bar: 10 µm.

• Supplemental Figure S2AB
• Supplemental Figure S2CD -

  Fig. S2. The MS/MS spectra of phosphopeptides acquired with a LTQ mass spectrometer. (A) Peptide of Nedd1 phosphorylated by Cdk1. (B-D) Peptides of Nedd1 phosphorylated by Plk1. The fragment ions are indicated as b-ions (red) and y-ions (blue), respectively. The phosphorylation sites in peptide sequences are marked with ‘#’. In B, the signal intensity is magnified fourfold in the m/z range from 200 to 750 and from 850 to 1550, respectively.

• Supplemental Figure S3 -

  Fig. S3. The sequential phosphorylation assay of the mutants of Nedd1 (341-end) by Cdk1 and Plk1. His-tagged Nedd1 (341-end) WT, Nedd1 (341-end) S411A, Nedd1 (341-end) S426A and Nedd1 (341-end) 4A were first incubated with or without Cdk1 for 30 minutes at 30°C, in the presence of nonradioactive ATP. After inhibition of Cdk1 by roscovitine, the Nedd1 proteins were further incubated with or without Plk1 in the presence of γ-³²PATP. After 30 minutes, the reactions were stopped and analyzed by SDS-PAGE followed by autoradiography (top). Coomassie Blue staining showed the loading amounts of Nedd1 proteins (bottom).

• Supplemental Figure S4 -

  Fig. S4. Characterization of the endogenous Nedd1 specific depletion and the Tet-on HeLa cell lines. (A) HeLa cells transfected with RNAi-insensitive GFP-Nedd1 WT, GFP-Nedd1 4A, GFP-Nedd1 4E, GFP-Nedd1 T550A or GFP-Nedd1 T550E were treated with Nedd1 RNAi or left untreated. The cell lysates were blotted with anti-Nedd1 and anti-γ-tubulin. The positions of the endogenous (Endo) Nedd1 and GFP-Nedd1 are indicated by arrows. (B) Tet-on HeLa cells stably transfected with RNAi-insensitive pcDNA4/TO-GFP-Nedd1 WT, pcDNA4/TO-GFP-Nedd1 4A, pcDNA4/TO-GFP-Nedd1 4E, pcDNA4/TO-GFP-Nedd1 T550A or pcDNA4/TO-GFP-Nedd1 T550E were cultured with or without tetracycline for 24 hours. The cell lysates were blotted with anti-Nedd1. The positions of the endogenous (Endo) Nedd1 and GFP-Nedd1 are indicated by arrows.