384 well white opaque plate

To measure intracellular cAMP, the AlphaScreen cAMP assay (Perkin Elmer) was used according to the manufacturer’s instructions. Briefly, CXCR1- and CXCR2-transfected HEK-293T cells (vide supra) were harvested and washed with PBS prior to being resuspended at final concentrations of 10⁷ cells per ml in freshly prepared stimulation buffer composed of 1 × ‘Hanks’ Balanced Salt Solution’ (HBSS; Lonza) complemented with 0.1 % (w/v) BSA, 0.5 nM IBMX (Sigma-Aldrich) and 5 mM HEPES (Lonza). Per well, 10⁴ cells were plated in a 384-well white opaque plate (Perkin Elmer) with 0.2 units/μL anti-cAMP acceptor beads in 5μL volume, to which 5μL of 10 μM forskolin and serial chemokine dilutions were added. After 30 minutes incubation at RT in the dark, 15μL of lysis buffer containing 1 Unit of streptavidin-labeled donor beads and 1 Unit biotinylated cAMP were added and the plate was incubated at RT in the dark for 1 h. The luminescence of the beads was read on a Synergy H4 plate reader through a 570/100 nm filter after sample excitation with 680/30 nm filtered light.

Interaction between GFP-β-DGNLS and ezrin or IMPα2/β1 heterodimer was assessed using an established ALPHAScreen assay (Perkin Elmer, Wellesley,MA) [21] (link), whereby IMPs α2 and β1 were pre-dimerized at 13.6 µM for 15 min at room temperature in intracellular buffer (20 mM HEPES pH 7.4, 110 mM KCl, 5 mM NaHCO₃, 5 mM MgCl₂, 1 mM EGTA, 0.1 mM CaCl₂, 1 mM DTT). Briefly, 30 mM of His₆-tagged GFP-β-DG-NLS fusion protein per well in a 384-well white opaque plate (Perkin Elmer) were incubated for 30 min with increasing concentrations of GST-ezrin or GST-IMPs (0–60 nM). 1 µl of a 1∶10 dilution of Nickel chelate acceptor beads and 1 µl of 2.5% BSA were added and incubated at room temperature for 90 min, followed by addition of 1 µl of a 1∶10 dilution of the streptavidin donor beads and incubation at room temperature for 2 h. Analysis was carried out using a FusionAlpha plate reader (Perkin Elmer).