![Figure 5. Identification of a CKIδ phosphorylation site on EB1. (A) A table summarizing the experimental conditions and results for assay 1. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows purified recombinant GST-EB1 used in the assay. Molecular mass (MM) is indicated in kilodaltons. (B) A table summarizing the experimental conditions and results for assay 2. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows the purified recombinant EB1 used in the assay. (A and B) Fragmentation spectra of the triply charged [M + 3H]3+ peptide ion at m/z 700.3676 (A) and m/z 700.367 (B) correspond to the phosphorylated peptide of aa 131–150 with the sequence phospho-QGQETAVAPSLVAPALNKPK of EB1. The predominant y-ion series for both spectra, extending from y11 to y17, confirm the sequence ETAVAP-phosphoS (dots). The fragment ions observed at m/z 667.93 (A) and 668.14 (B) correspond to the neutral loss of H3PO4 from the precursor ions, whereas those observed at m/z 662.14 (A) and 662.14 (B) correspond to the neutral loss of H3PO4 and H2O. GST-CKIδ and CKIδ[1–318] were commercially sourced. (C) A schematic diagram shows the position of the phosphorylation site in EB1. The domains shown are calponin homology (CH) and end binding homology (EBH).](https://cdn.rupress.org/rup/content_public/journal/jcb/195/5/10.1083_jcb.201106025/5/jcb_201106025_gs_fig5.jpeg?Expires=1771602306&Signature=GQWuE1upSCQeokKvk73Z2zAi7R0GEN0xywekgzParufYwVkqeIcqZ09xeNNtuGq3K42zrBmluR4tj603bJuJpy1QAzhhGwfluc1oFckL0xGshFCyOuT374kZutOsLwiBpvS3KDsI6U9e6Qc1ViKldtuZQG3kfRWrv~auqiKtBNXkGv-QV~afcBqDsT7X-2gi0k2bOeRFPD-pNSFPNEBMx9xuCkPgY5PtsE~9flDnJQcCBgl7cXMbWkYlCRRJymB107fvyRSz6Zsm1v-DulGTNNQxtZA~DKufKMTdJaEft9L~ABMPpkNZdnqkVVf~taEfb-vVV09DVbjTAGXxPmyT9w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Identification of a CKIδ phosphorylation site on EB1. (A) A table summarizing the experimental conditions and results for assay 1. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows purified recombinant GST-EB1 used in the assay. Molecular mass (MM) is indicated in kilodaltons. (B) A table summarizing the experimental conditions and results for assay 2. Fragmentation spectra were obtained and analyzed for all conditions. The spectrum on the right corresponds to 10 units of kinase. Ponceau S staining shows the purified recombinant EB1 used in the assay. (A and B) Fragmentation spectra of the triply charged [M + 3H]3+ peptide ion at m/z 700.3676 (A) and m/z 700.367 (B) correspond to the phosphorylated peptide of aa 131–150 with the sequence phospho-QGQETAVAPSLVAPALNKPK of EB1. The predominant y-ion series for both spectra, extending from y11 to y17, confirm the sequence ETAVAP-phosphoS (dots). The fragment ions observed at m/z 667.93 (A) and 668.14 (B) correspond to the neutral loss of H3PO4 from the precursor ions, whereas those observed at m/z 662.14 (A) and 662.14 (B) correspond to the neutral loss of H3PO4 and H2O. GST-CKIδ and CKIδ[1–318] were commercially sourced. (C) A schematic diagram shows the position of the phosphorylation site in EB1. The domains shown are calponin homology (CH) and end binding homology (EBH).
