Enzymes, Signaling Proteins, Antibodies

These results are consistent with the large thermal shift observed in the initial compound library screen . HA-cyclin Y WT was co-transfected with FLAG-CDK16 WT or D304A kinase-inactive mutant in COS1 cells as indicated and cells were treated for 1 h with 10 μM of the indicated inhibitors . Cell lysates were immunoprecipitated with FLAG-agarose and immunoblotted using the indicated antibodies. HA-cyclin Y WT or S336A mutant was co-transfected with FLAG-CDK16 WT or D304A kinase-inactive mutant in COS1 cells as indicated. Cells were treated for 1 h with varying concentrations of the indicated inhibitors, and cell lysates were immunoblotted using the indicated antibodies. FLAG immunoblot is representative of all three inhibitors.
On the other hand, we found that PCTK3 bound to cyclin A2 in the cytoplasm and promoted the translocation of cyclin A2 from the nucleus to the cytoplasm. These results demonstrated that PCTK3 is a cytoplasmic binding partner of PCTAIRE Antibodies cyclin A2 and that cyclin A2 alters its subcellular localization by binding to different partners. Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes.

Avantor® can help equip your life sciences lab with the products, equipment, and supplies you need – whether you work in cell biology, genomics, proteomics, or other fields. ELISA Protocol Troubleshooting and FAQs Antibody FAQs Storage Upon receipt, store at -20°C or -80°C. Lead Time Basically, we can dispatch the products out in 1-3 working days after receiving your orders. Delivery time maybe differs from  different purchasing way or location, please kindly consult your local distributors for specific delivery time. When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
The fact that transformed cells display high levels of PCTAIRE-1 protein suggests that the kinase is likely to be involved in proliferation. Contrary to cdk5, which is widely expressed but displays kinase activity only in neuronal cells , PCTAIRE-1 appears to be active in both normal and transformed cells . We have identified a direct interaction between the COPII machinery and a ubiquitously expressed protein kinase family, PCTAIRE. Furthermore, we have shown that manipulation of PCTAIRE kinase activity modulates membrane trafficking through the early secretory pathway. These findings are of particular interest for a number of reasons. First, there is considerable evidence for protein kinase regulation of membrane traffic at the ER-Golgi interface.

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Nuclear localization conferred by the pocket domain of the retinoblastoma gene product. The cyclin box and C terminus of cyclins A and E specify CDK activation and substrate specificity. A role for regulated binding of p150 to microtubule plus ends in organelle transport. Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.

P57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Fabbro, D., Batta, D., Rose, P., Schacher, B., Roberts, T. M., and Ferrari, S. Homogeneous purification of human recombinant GST-Akt/PKB from Sf-9 cells. Shin B. C., Suzuki M., Inukai K., Anai M., Asano T., Takata K. Multiple isoforms of the regulatory subunit for phosphatidylinositol 3-kinase (PI3-kinase) are expressed in neurons in the rat brain. Michaelis C., Weeks G. The isolation from a unicellular organism, Dictyostelium disciodeum, of a highly-related cdc2 gene with characteristics of the PCTAIRE subfamily.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human PCTAIRE 1. Antibodies are purified by protein A and peptide affinity chromatography. For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. These tissue lysates can be used in applications such as SDS-PAGE and Western blotting. Whole cell lysates can be used as positive controls for applications such as ELISAs, immunoprecipitation and Western blotting. Obtaining high-quality whole cell lysates can be tedious as well as time consuming.
The third member of this family, PCTK3, has been the least studied. Although exogenously expressed PCTK1 and PCTK2 phosphorylate myelin basic protein and histone H1 in vitro, PCTK3 kinase activity has not been detected. To characterize PCTK3 activity and elucidate its regulatory mechanism, it is necessary to identify activators for PCTK3. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport.

Upon entry into S phase, PCTAIRE-1 activity increased rapidly, displaying a peak during the transition through the S phase and G2 phase. The overall level of endogenous PCTAIRE-1 protein did not change during progression through the cell cycle (Fig. 4,C). To exclude cell type-specific effects or artifacts due to the method of synchronization used, we examined PCTAIRE-1 kinase activity in a different system. HeLa cells were arrested by double thymidine block, and the extent of synchronization upon release from the block was examined by flow cytometric analysis of DNA content (Fig. 5,A). The high synchronicity of the system was confirmed by Western blot analysis of cyclin A (Fig. 5,B) and cyclin B1 (Fig. 5,C) expression.
A weak association was also seen with Sec24Dp but not with lamin or Sec13p. The weakly detectable interaction with Sec31Ap in plate growth assays was not seen in complementary colorimetric assays and therefore was deemed a false positive. The two-hybrid clone encoding PCTAIRE-3 includes nine amino acids from predicted intron c and 110 amino acids from predicted intron h. Using PCR, we generated a `fully-spliced' cDNA including exons 4-8, which is equivalent to the central region of the predominant isoform of PCTAIRE-3, PCTAIRE-3a (Herskovits and Davies, 2004; Okuda et al., 1992). The amino acids encoded by predicted intron h are believed to arise from incomplete splicing as they include a stop codon after 61 amino acids. The nine amino acids included in this clone would encode a known splice form, PCTAIRE-3b .

Considering that CCNY is a shared cyclin partner of atypical CDKs, CCNY may regulate Wnt signaling by phosphorylating LRP5/6 together with CDK14 , which further reflects the complexity of cyclin-CDK function. Direct binding assays showing an interaction between PCTAIRE-1 and Sec23Ap, although reproducible, consistently showed very weak binding of the two components. This might reflect a low affinity interaction or the fact that there is some regulation of this interaction. Alternatively, some unknown component may be required to increase the affinity of binding, or even to facilitate the interaction. This would explain the more reliable co-immunoprecipitation of these two components from cell lysates in which additional factors would be present.
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The hydroxybutyloxime moiety extends across the expected ribose- and phosphate-binding sites of the ATP  pocket. Here, the flipped-out conformation of Phe305 in the activation segment establishes a cage-like structure around the inhibitor. The binding therefore features an induced fit that maximises the kinase–inhibitor interaction. For the rebastinib complex, CDK16 protein was buffered in 25 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol and 10 mM DTT.