ProtocolCell Biology

A Dual Array-Based Approach to Assess the Abundance and Posttranslational Modification State of Signaling Proteins

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Science Signaling  10 Jan 2012:
Vol. 5, Issue 206, pp. pl1
DOI: 10.1126/scisignal.2002372


  • Fig. 1

    Monitoring β-catenin by bead array assay or lysate array. (A) The β-catenin bead array panel uses antibodies and known interaction partners of β-catenin to study the phosphorylation status and its complexation status within the same multiplex assay system. Different amounts of protein extracts from untreated HepG2 cells were analyzed. The results are given in median fluorescence intensities (MFI). (B) A schematic of lysate microarrays (reverse-phase protein microarrays). In lysate microarrays, samples are immobilized on a nitrocellulose-coated slide and each slide is probed with a different antibody. The microarray format enables several thousand samples to be printed on a single slide, and multiplexing is achieved by printing many different copies of the same array.

  • Fig. 2

    Snapshots of different forms of β-catenin monitored by bead array assay. HepG2 cells were starved for 24 hours and then treated with 200 ng/ml recombinant Wnt3a (filled squares) or recombinant Wnt5a (open circles) for 5, 30, 60, 180, 300, and 1200 min. Each experiment was performed in biological triplicates. From each sample, 25 μg was analyzed by using the β-catenin bead array panel. Mean and standard deviation of fold-change were calculated from three independent experiments and are displayed as ratios (treated/untreated), including the corresponding error propagation.

  • Fig. 3

    Monitoring the dynamic changes in proteins in response to Wnt ligands in HepG2 cells using lysate microarrays. (A) Wnt3a and Wnt5a stimulate opposite changes in cellular content of the canonical Wnt signaling components, β-catenin, axin2, and TCF1. Both Wnt3a and Wnt5a stimulation caused similar changes in the phosphorylation of extracellular signal regulated kinase (pERK). (B) Heat maps showing changes in total levels of 10 proteins and phosphorylation status of 11 signaling proteins upon Wnt3a and Wnt5a stimulation. (C) A knowledge-based pathway diagram based on the heat plot (B) showing opposite effects of a canonical Wnt ligand (Wnt3a) and a noncanonical ligand (Wnt5a) in HepG2 cells. Green represents proteins (or phosphorylated proteins) that showed an increase, red represents proteins that showed a decrease, purple represents proteins that were measured but did not change, and gray represents proteins that were not measured.


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