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Quantitative analysis of the proteome of tankyrase double knockout cells using isobaric tandem mass tags reveals targets of degradation, including antagonists of the Wnt/β-catenin signaling pathway (NKD1, NKD2, and Hect D1) and three (Notch 1, 2, and 3) of the four Notch receptors.
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We observed the most robust effect (stabilization of Axin1 and loss of β-catenin) in the DKO cell line, indicating it as the optimal line to use for identifying targets of tankyrase-mediated degradation. a Immunoblot of HEK293T TNKS KO cell lines demonstrating stabilization of Axin1 and reduction of β-catenin in TNKS KO cell lines.
Protein levels of Axin and β-catenin in total cell lysates).
To analyze telomere cohesion, we isolated cells by mitotic shake-off and subjected them to FISH analysis with a sub telomere specific probe 16p (Fig.
DKO cells revealed an even greater level (small, but significant), of persistent cohesion than the single KOs.
Considering that Notch signaling is commonly activated in cancer, tankyrase inhibitors may have therapeutic potential in targeting this pathway..
The preliminary success of these drugs has led to an interest in targeting other members of the PARP family.
Tankyrase protein was analyzed in the KO cell lines using immunoblot analysis with antibodies raised against tankyrase 1 (that also cross-react weakly with tankyrase 2)Generation and functional analysis of human tankyrase knockout cell lines.
a Immunoblot analysis of whole cell lysates from HEK293T knockout (KO) cell lines generated by CRISPR-Cas9 stained with antibody that detects tankyrases 1 and 2. The role of tankyrase 2 in this process has not been determined.
In addition, we performed a quantitative analysis of the proteome in tankyrase double knockout cells and report on targets of tankyrase–mediated proteasomal degradation.
KO clones were generated (Supplementary Figure 1 a, b).
The role of tankyrase 2 in this process has not been determined.