Journal: Leukemia

Article Title: Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation.

doi: 10.1038/sj.leu.2402793

Figure Lengend Snippet: Figure 1 Akt1 activity in HL60PT or AR cells. Western blot analy- sis for expression of total Akt1 and p-Akt1 forms (a). For inhibition with Ly294002 (Ly, 5 M) or wortmannin (wort, 0.1 M) cells were incubated in the presence of the inhibitors for 24 h. 80 g of protein was blotted to each lane. The blots are representative of at least three separate experiments. (b) In vitro Akt1 kinase activity. For inhibition with Ly294002 (Ly) or wortmannin (wort) cells were incubated in the presence of the inhibitors for 24 h. The results are the mean ± s.d. of three separate experiments.

Article Snippet: The following primary antibodies were used in this study: from Upstate Biotechnology: rabbit polyclonals to Akt1, cytochrome c, mouse monoclonals to human c-myc (clone 9E10) and caspase-9; from Santa Cruz Biotechnology (Santa Cruz, CA): mouse monoclonals to NF- B p50 subunit and FLICEinhibitory protein (cFLIPS/L); from Cell Signaling Technology (Beverly, MA, USA): mouse monoclonals to PTEN and BID, rabbit polyclonals to caspase-10, Thr 308 phosphorylated Akt1 (p-Akt1), Ser 473 p-Akt1, Bcl-2, Bcl-XL; from Sigma: mouse monoclonal to -tubulin, rabbit polyclonal to procaspase-8; from R&D Systems (Oxon, UK): goat polyclonals to extracellular domain of human DR4, DR5, DcR1, DcR2; from Molecular Probes (Leiden, The Netherlands): rabbit polyclonal to cytochrome oxidase subunit II (COX II).

Techniques: Activity Assay, Western Blot, Expressing, Inhibition, Incubation, In Vitro

Journal: Leukemia

Article Title: Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation.

doi: 10.1038/sj.leu.2402793

Figure Lengend Snippet: Figure 6 Overexpression of human PTEN sensitizes HL60AR cells to TRAIL. (A) Western blot analysis for PTEN expression in HL60AR, mock-transfected HL60AR cells (AR MT) and in three stably trans- fected clones. 80 g of protein was blotted to each lane. The blot is representative of at least three separate experiments. (B) Levels of in vivo synthesized PtdIns (3,4,5)P3 in HL60AR cells and in the three stably transfected clones overexpressing PTEN. A representative auto- radiograph from three separate experiments is shown. In this experi- ment, incorporation was 1987 c.p.m. for AR cells, 604 c.p.m. for clone 4H1, 559 c.p.m. for clone 4H9 and 517 c.p.m. for clone 7E5. (C) In vitro PTEN activity in HL60AR, AR MT cells, and in three stably transfected clones; 100% of the activity was 257.1 ± 21.4 pmoles P released/reaction. (D) In vitro Akt1 kinase activity in HL60AR, AR MT cells, and in three stably transfected clones; 100% of the activity was 4087 ± 356 c.p.m. incorporated/reaction. (E) Flow cytometric analysis for TRAIL-induced apoptosis (500 ng/ml TRAIL for 6 h) in HL60AR, AR MT cells, and in three stably transfected clones. In c,d, e, the results are the mean ± s.d. of three separate experiments.

Article Snippet: The following primary antibodies were used in this study: from Upstate Biotechnology: rabbit polyclonals to Akt1, cytochrome c, mouse monoclonals to human c-myc (clone 9E10) and caspase-9; from Santa Cruz Biotechnology (Santa Cruz, CA): mouse monoclonals to NF- B p50 subunit and FLICEinhibitory protein (cFLIPS/L); from Cell Signaling Technology (Beverly, MA, USA): mouse monoclonals to PTEN and BID, rabbit polyclonals to caspase-10, Thr 308 phosphorylated Akt1 (p-Akt1), Ser 473 p-Akt1, Bcl-2, Bcl-XL; from Sigma: mouse monoclonal to -tubulin, rabbit polyclonal to procaspase-8; from R&D Systems (Oxon, UK): goat polyclonals to extracellular domain of human DR4, DR5, DcR1, DcR2; from Molecular Probes (Leiden, The Netherlands): rabbit polyclonal to cytochrome oxidase subunit II (COX II).

Techniques: Over Expression, Western Blot, Expressing, Transfection, Stable Transfection, Clone Assay, In Vivo, Synthesized, In Vitro, Activity Assay

Journal: Leukemia

Article Title: Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation.

doi: 10.1038/sj.leu.2402793

Figure Lengend Snippet: Figure 7 Overexpression of constitutively active Akt1 renders HL60PT cells resistant to TRAIL. (A) Western blot analysis for Akt1 expression in HL60PT, mock-transfected HL60PT cells (PT MT), and in three stably transfected clones. 80 g of protein was blotted to each lane. Overexpressed Akt1 was revealed by means of a monoclonal antibody to the myc-tag. The blot is representative of at least three separate experiments. (B) In vitro Akt1 kinase activity in HL60PT, PT MT cells, and in three stably transfected clones; 100% of the activity was 4234 ± 378 c.p.m. incorporated/reaction. (C) Flow cytometric analysis of TRAIL-induced apoptosis (2 g/ml TRAIL for 6 h) in HL60PT, PT MT cells, and in three stably transfected clones. In (B) and (C) the results are the mean ± s.d. of three separate experiments.

Article Snippet: The following primary antibodies were used in this study: from Upstate Biotechnology: rabbit polyclonals to Akt1, cytochrome c, mouse monoclonals to human c-myc (clone 9E10) and caspase-9; from Santa Cruz Biotechnology (Santa Cruz, CA): mouse monoclonals to NF- B p50 subunit and FLICEinhibitory protein (cFLIPS/L); from Cell Signaling Technology (Beverly, MA, USA): mouse monoclonals to PTEN and BID, rabbit polyclonals to caspase-10, Thr 308 phosphorylated Akt1 (p-Akt1), Ser 473 p-Akt1, Bcl-2, Bcl-XL; from Sigma: mouse monoclonal to -tubulin, rabbit polyclonal to procaspase-8; from R&D Systems (Oxon, UK): goat polyclonals to extracellular domain of human DR4, DR5, DcR1, DcR2; from Molecular Probes (Leiden, The Netherlands): rabbit polyclonal to cytochrome oxidase subunit II (COX II).

Techniques: Over Expression, Western Blot, Expressing, Transfection, Stable Transfection, Clone Assay, In Vitro, Activity Assay