We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 2

Figure 1

Figure 1. Preparation of Xenopus egg extract for HTS screening. From: Screening for small molecule inhibitors of embryonic pathways: Sometimes you gotta crack a few eggs.

Xenopus females are injected with human chorionic gonadotropin to induce egg laying into containers filled with buffer. Eggs are collected and the jelly coat removed by treatment with 2% cysteine. The dejellied eggs are washed and subjected to a packing spin (~100 × g) to remove excess buffer. The packed eggs are then subjected to a crushing spin (>15,000 × g) that separates the crushed egg components into three distinct layers: lipid, cytoplasmic, and pigmented granule/yolk. The cytoplasmic layer is collected and can be used for biochemical studies or for HTS. Recombinant proteins can be added directly to the egg extract; alternatively, if recombinant proteins are not readily available, mRNA encoding the desired proteins can be added instead. For the latter, the egg extract must be freshly prepared because frozen extract loses its capacity to translate mRNAs. To immunodeplete specific proteins from extract, antibodies linked to resin can be added and removed. Finally, a specific compound can be added to perturb the signaling pathway followed by screening for other compounds that either synergize with it or block its effects. Screen readouts may include fluorescence with GFP constructs, luminescence with luciferase constructs, or radioactivity using scintillation proximity assays. Microsopic analysis of cellular structures (e.g. mitotic spindle, nuclear envelope, chromosomes, etc.) can also be performed in a high throughput manner with appropriate imaging analysis software.

Brian I. Hang, et al. Bioorg Med Chem. ;20(6):1869-1877.
Figure 2

Figure 2. Schematic of the Wnt, Hedgehog, and Notch signaling pathways highlighting cytoplasmic degradation of key transcriptional mediators. From: Screening for small molecule inhibitors of embryonic pathways: Sometimes you gotta crack a few eggs.

For Wnt signaling, the transcriptional coactivator, β-catenin, is degraded in the absence of a Wnt signal, whereas the scaffold protein, Axin, is stable. Binding of Wnt ligands to the Frizzled and LRP5/6 coreceptors results in inhibition of β-catenin degradation whereupon it enters the nucleus. Transcription factors such as TCF, BCL9, and Pygopus form a complex with nuclear β-catenin that leads to activation of a Wnt transcriptional program. In contrast, Axin degradation is stimulated upon Wnt pathway activation. Members of the GLI family of transcriptional factors are the mediators of canonical Hedgehog (HH) signaling. In the absence of HH, GLI proteins are degraded or converted to a lower molecular weight form (GLI-R), which acts as a transcriptional repressor. Proteolysis of GLI occurs in a cytoplasmic complex containing several kinases including glycogen synthase kinase 3 (GSK3), casein kinase 1α (CK1α), and protein kinase A (PKA). The binding of HH to its receptor, Patched (Ptch), relieves inhibition of the seven membrane-spanning protein, Smoothened (Smo), by Ptch via an unknown mechanism. The uninhibited Smo protein subsequently inhibits GLI proteolysis, promoting accumulation of the full-length form and subsequent activation of HH target genes. Notch signaling is initiated upon binding of the transmembrane Notch protein to the Delta/Serrate/LAG-2 (DSL) family of plasma transmembrane ligands present in the membrane of adjacent cells. Binding results in a series of proteolytic cleavage events that ultimately release the Notch intracellular domain (NICD) into the cytoplasm followed by its translocation into the nucleus. In the absence of NICD, the CBF1/Suppressor of Hairless/LAG1 (CSL) family of DNA binding proteins associates with corepressors (CoR) to inhibit Notch target gene transcription. Nuclear NICD interacts with CSL and the transcriptional coactivator Mastermind (MAML1) to recruit transcriptional coactivators (CoA) to initiate transcription of Notch target genes.

Brian I. Hang, et al. Bioorg Med Chem. ;20(6):1869-1877.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk