recombinant human akt1 protein  (Novus Biologicals)

Journal: Cancer discovery

Article Title: AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B

doi: 10.1158/2159-8290.CD-20-0815

Figure Lengend Snippet: A. IB analysis of indicated protein levels in PC-3 cells were treated with DMSO or indicated compounds at 1 μM for 24 hr. B-C. (B) Cell cycle analysis by flow cytometry of PC-3 cells after 48 hr treatment with DMSO, AZD5363, MS21, or GDC-0941. Histograms are representative of triplicates. (C) Quantification of cell cycle phases of PC-3 cells after 48 hr treatment as indicated. Percentages reflect the mean of triplicates, and error bars indicate SEM. D. Indicated cells were treated with DMSO or MS21 at 0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM for 2 weeks. Cells were stained with Crystal Violet. E. In vitro AKT/PKB phosphorylation of AURKB. Recombinant active full-length human AKT1/PKB protein (400 ng) and recombinant active full-length human AURKB protein (500 ng) were incubated with or without 200 μM ATP in 50 μL 1 × kinase buffer at 30°C for 30 min. 600 μM of Barasertib or AZD5363 were pre-incubated with recombinant protein for 20 min before ATP was added into the reactions. IB analysis of phosphorylation of AURKB using anti-Phospho-AKT Substrate (RXXS*/T*) antibody after kinase assay. F. IP of HA-AURKB protein and IB analysis of phospho-AKT substrate in 293T-AURKB-T232A or 293T-AURKB-T73A/T232A cells treated with Insulin (200 μg/mL) for 0, 10, 30, 60 mins after starvation for overnight. G. IP of HA-AURKB and IB analysis of phosphor-AKT substrate in 293T-HA-AURKB cells pretreated with or without GDC0941 (1 μM) for 10 mins and followed by insulin (10 μg/mL) stimulation for 10 mins. H. IP of HA-AURKB and IB analysis of phospho-AKT substrate in PC-3-AURKB cells treated with DMSO or AZD5363 (1 μM) for 2 hr. I. IP of endogenous AURKB and IB analysis of phospho-AKT substrate in PC-3 cells pretreated with or without AZD5363 (1 μM) for 20 mins and followed by insulin (200 μg/mL) stimulation for 10 mins. J. Indicated cells were treated with DMSO or MS21 at 3 μM for 24 hr and lysates tested for AURKB, T-AKT. K. IB analysis of AURKB protein in PC-3 cells treated with GDC0941 (1 μM) and MG132 (10 μM) for indicated times.

Article Snippet: AKT kinase assay: Recombinant active full-length human Akt1/PKB protein (400 ng) (Cat#14–276, Millipore Sigma) was incubated with 200 μM ATP (Cat#20–306, Sigma) and 500 ng recombinant active full-length human AURKB protein (Cat#14–835, Millipore Sigma) in 50 μl 1 × kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, Cat#9802, Cell Signaling Technology).

Techniques: Cell Cycle Assay, Flow Cytometry, Staining, In Vitro, Phospho-proteomics, Recombinant, Incubation, Kinase Assay

Journal: Cancer discovery

Article Title: AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B

doi: 10.1158/2159-8290.CD-20-0815

Figure Lengend Snippet: A. Summary of the sensitivity to MS21 treatment in the cell lines tested by colony formation assay, and status of KRAS mutations, BRAF mutations, PIK3CA mutations, PTEN mutations, HER2+ status, AKT1 mutations and tissue origin of the cell lines. Cells are categorized into two groups: resistant and sensitive. Red asterisk label indicates cell lines which are more sensitive to AKT degrader MS21 than AZD5363. B. Cellular activity of AZD5363 and MS21 across a panel of indicated cell lines measured as the effects on cell viability tested and quantified in colony formation assay. Grey line indicated AZD5363 treatment and black line indicated MS21 treatment. C. Analysis of cell lines that are sensitive or resistant to MS21 treatment based on their mutation status on the PI3K/PTEN pathway and Ras pathways. Fisher’s exact test was performed to identify any enrichment of sensitive or resistant cell lines between mutated and wide type cell lines. Cell numbers are shown with white color on the columns. D. IB analysis of indicated protein levels in the indicated cells treated with DMSO or MS21 for 24 hr. Blue characters indicate sensitive cell lines and red characters indicate resistant ones. E. Lysates from PC-3 and MiaPaca2 cells were treated with DMSO or MS21 at 1 μM for 4 hr at the indicated temperatures and then analyzed by immunoblot with T-AKT and β-actin antibodies.

Article Snippet: AKT kinase assay: Recombinant active full-length human Akt1/PKB protein (400 ng) (Cat#14–276, Millipore Sigma) was incubated with 200 μM ATP (Cat#20–306, Sigma) and 500 ng recombinant active full-length human AURKB protein (Cat#14–835, Millipore Sigma) in 50 μl 1 × kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, Cat#9802, Cell Signaling Technology).

Techniques: Colony Assay, Activity Assay, Mutagenesis, Western Blot