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IC50 Values from ICW Assays Correlate Well with Published Values

Method Parameter measured Result
In-Cell Western CREB ap pKB 8.72±0.13
cAMP cAMP ap pKB 8.72±0.07
Radioligand binding assay Binding pK 8.40±0.05

Table 1. pKi, the negative log of the inhibition constant of a drug; pKB, the negative log of the dissociation equilibrium constant for a competitive antagonist; ap, apparent; CREB, cAMP response element binding protein. Selkirk JV et al. J. Biomol. Screen. 11: 351 (2006).

Comparison to GPCR Activity Assay

GPCR activity assays primarily monitor upstream signaling events, such as accumulation of cyclic AMP (cAMP). Downstream events, such as phosphorylation of cAMP response element binding protein (CREB), may also be useful readouts.1-2

In a 2006 study, the In-Cell Western method was used to quantify CREB phosphorylation.1 Potencies of agonists and antagonists correlated well with radioligand binding affinities and cAMP accumulation assays.

For the GPCR antagonist KW6002, the results in Table 1 were obtained.

Additional Publications:

  1. Aguilar HN et al.
    Quantification of rapid myosin regulatory light chain phosphorylation using high-throughput In-Cell Western assays: comparison to Western immunoblots.
    PLoS ONE 5(4): e9965 (2010).
  2. Hoffman, GR et al.
    A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In-Cell Western technique.
    Assay Drug Dev Tech 8(2):186-99 (2010).
  3. Kumar N et al.
    Multipathway model enables prediction of kinase inhibitor cross-talk effects on migration of Her2-overexpressing mammary epithelial cells.
    Mol Pharmacol 73:1668-1678 (2008).
  4. Selkirk JV et al.
    A novel cell-based assay for G-protein-coupled-receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation.
    J Biomol Screen 11: 351 (2006).
  5. Chen H et al.
    A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.
    Anal Biochem 338:136-142 (2005).
  6. Wong SK-F.
    A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors.
    Anal Biochem 333:265-72 (2004).
  • 1. Selkirk JV et al. J Biomol Screen 11: 351 (2006)
  • 2. Wong SK-F. Anal Biochem 333:265-72 (2004)

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