Figure 1. Calcium release rapidly induces AurA auto-phosphorylation.

(a) Western blot assay confirming expression of the AVPR1 receptor in cell lines used in this study. MCF7 were used as a positive control for AVPR1 expression, based on North et al.60. (b) Immunofluorescence of HK-2 cells 30 s after stimulation with 100 nM arginine vasopressin (+AVP) versus control untreated cells (−AVP). Cells were visualized with antibodies to AurA (red) and T²⁸⁸-phospho-AurA (phAurA, green) and DAPI (blue), as indicated; scale bar, 20 μm. Insets show magnification of indicated centrosomes. (c) Analysis of data from b quantifies phAurA-positive cells at times after stimulation. (d) Analysis of data from b quantifies relative intensity of AurA in cells within 20 s after stimulation with AVP. (e) Western blot analysis of AVP-treated HEK293 cells overexpressing AurA in the presence or absence of extracellular Ca²⁺ chelator (5 mM EGTA) (f) Western blot analysis of phAurA in HEK293 cells transfected with an AurA-expressing plasmid 1–2 min after stimulation with 10 μM histamine. For each SDS–PAGE, β-actin is used as a loading control and molecular weight is indicated in kDa. For c and d, an average of 150 cells were counted. (c, d, e) N=3 with error bars indicating the standard error. *P<0.05.