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. 2016 Apr 19;7:11389. doi: 10.1038/ncomms11389

Figure 9. Aurora controls localization and phosphorylation of the tyrosine kinase Lck.

Figure 9

(a) Maximum Z projection of XYZ stack of human Jurkat T cells pretreated with vehicle (DMSO) or Aurora A inhibitor (MLN8237) and conjugated with SEE-preloaded Raji B cells (APCs; 30 min). Cells were fixed and stained for α-tubulin–fluorescein isothiocyanate (FITC) (green), PKCθ (magenta) and Lck (red). Bright-field images are included. Scale bar, 10 μm. (b) Quantification of Lck accumulation at the IS in conjugates as in a from three independent experiments (DMSO, n=96. MLN8237, n=94). Data represent means±s.d. Means were compared with a t-test; ****P<0.0001. (c) Immunoblotting of Lck phosphorylation at Y394 in primary human CD4+ T cells. Cells were pretreated with DMSO or MLN8237 and conjugated for the indicated times with anti-CD3/CD28-coated beads. Total Lck and actin are included as loading controls. Arrows point Lck band. (d) Quantification of data from four independent experiments as in c. Error bars represent s.d. Medians were compared with a Friedman test (*P<0.05). (e) Immunoblots of CD3ζ phosphorylation in lysates of J.CAM1 T cells transfected with Lck-GFP, CD4-Lck or CD4-ΔCyt-GFP, pretreated with DMSO or MLN8237 and conjugated for 5 min with SEE-pulsed APCs. (f) T-cell lymphoblasts pretreated with DMSO or MLN8237 were activated or not with SEE-pulsed APCs (2 min) and subjected to IP using an anti-Lck antibody. The immunoprecipitates were subjected to MS analysis. Upper panel, MS/MS extracted ion chromatograms of the Y394-phosphorylated and non-modified forms of Lck peptide LIEDNEYTAR. Lower panel, phosphorylated:non-modified peak ratios. (g) Recombinant Lck was incubated with Aurora A WT (in the absence or presence of MLN8237) or Aurora A KD immunoprecipitated from nocodazole-treated (16 h), transfected HEK293 cells. Lck and Aurora were incubated for 30 min in the presence of ATP and the mixture analysed by MS. Upper panel, MS/MS extracted ion chromatograms of the Y394-phosphorylated and non-modified forms of Lck peptide LIEDNEYTAR. Lower panel, phosphorylated:non-modified peak ratios. See Supplementary Table 1 for representative MS/MS spectra of the phosphorylated and non-phosphoryated forms of the peptide at the peaks.