Figure 5.

DIDO Associates with and Is Phosphorylated by PKCiota

(A) PKCiota phosphorylation of DIDO peptide MDDKGHLSNEEAPK on residue Ser8. Top: electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) spectrum of the m/z 551.1 ion corresponding to the phosphorylated peptide; pS indicates a phosphorylated serine residue. Bottom: ETD MS/MS spectrum of the m/z 524.5 ion corresponding to the non-phosphorylated peptide. Peptide sequences are shown with the identified c-/z-type ions.

(B) Left: anti-HA immunoprecipitates of a negative control, WT HA-DIDONT, HA-DIDONTSer8Ala, shortened HA-DIDONT, and anti-aPKC as positive control were used as substrates in an in vitro kinase assay with recombinant PKCiota and zeta; autoradiograph shows [³²P]ATP incorporation (arrowheads). Right: western blot controls of lysates and precipitates of corresponding proteins.

(C) Labeling of centrosomes (arrowheads) in WT 2-cell stage rosettes with γ-TUBULIN (red) and DIDO MAB-1C6 (green; nuclear only).

(D) Labeling of centrosomes (arrowheads) in WT 2-cell stage rosettes with PAB-DIDO3 (green) and γ-TUBULIN (red).

Scale bars: (C and D) 10 μm.