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

Abstract

A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of β-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of β-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.

Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells

“…4a), multiplex arrays were applied for detailed status analysis of the WNT pathway effector β-catenin3233. A first response was readily detected after 2 h (120 min) post CHIR exposure (Supplementary Fig.…”

“…Supernatants were analysed by bead array-based sandwich immuoassays for total β-catenin, β-catenin phosphorylated at Ser33/ Ser37/Thr41, β-catenin phosphorylated at Ser45, β-catenin phosphorylated at Ser552 and β-catenin phosphorylated at Ser 675 as described earlier32. Free β-catenin was investigated by bead array-based glutathione S -transferase-pull-down assays using glutathione S -transferase fusion proteins for β-catenin and Tcf (ICAT), transcription factor 4 (TCF4) and the cytoplasmic domain of E-cadherin as baits3233.…”

Suppression of Host p53 Is Critical for Plasmodium Liver-Stage Infection

“…Both Akt antibodies have been previously validated in a variety of cell lines, including HepG2 cells (Luckert et al, 2012). Encouragingly, signal for all four antibodies in the Akt/mTor pathway recorded increases in response to parasite infection (Figure S1C).…”

“…Cells were treated with our without Nutlin-3 as describe above. At the desired time point, cells were harvested and stained as described previously (Luckert et al, 2012). All experimental conditions were tested in biological triplicate.…”

Co-clinical assessment identifies patterns of BRAF inhibitor resistance in melanoma

“…Key advantages of RPPA and other protein array technologies (40,41) are their relatively low cost per sample and rapid turnaround. Indeed, RPPA is already being used to direct clinical trials (NCT01074814, NCT01919749, and NCT02008994; https://clinicaltrials.gov/) in a clinically relevant time frame.…”