Journal:

Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

doi: 10.1128/MCB.24.23.10289-10299.2004

Figure Lengend Snippet: Post-cytochrome c protection by Bcr-Abl in Xenopus egg extracts. (A) Crude egg extract was incubated with buffer alone, WT Bcr-Abl, or kinase-dead (K671R) Bcr-Abl, and caspase 3 activity was measured at various time points. Caspase 3 activity was measured spectrophotometrically via cleavage of the colorimetric caspase substrate DEVD-pNA. (B) Cytosolic egg extract was pretreated for 30 min with WT Bcr-Abl, K671R Bcr-Abl, or buffer alone and then incubated with 0.6 ng of cytochrome c per μl. Caspase 3 activity was measured by cleavage of DEVD-pNA over time. (C) Cytosolic egg extract was pretreated for 30 min with WT or K671R mutant Bcr-Abl and then incubated with 0.7 ng of cytochrome c per μl for various times. Caspase 9 activity was measured spectrophotometrically by cleavage of the colorimetric caspase 9 substrate LEHD-pNA. (D) In vitro-translated, 35S-labeled procaspase 9 and soluble cytochrome c (0.6 ng/μl) were added to cytosolic egg extracts that were pretreated with WT or K671R mutant Bcr-Abl. Cleavage of procaspase 9 was assessed via autoradiography. In panels A to C, the graphs are representative of at least three independent experiments. OD405, absorbance reading at 405 nm.

Article Snippet: Human procaspase 9 was immunoprecipitated from human embryonic kidney 293T cells with polyclonal caspase 9 antiserum (2 μg of antibody per mg of total protein; BD Biosciences Pharmingen).

Techniques: Incubation, Activity Assay, Mutagenesis, In Vitro, Labeling, Autoradiography

Journal:

Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

doi: 10.1128/MCB.24.23.10289-10299.2004

Figure Lengend Snippet: Bcr-Abl prevents processing of caspase 3 and caspase 9. (A) In vitro-translated, 35S-radiolabeled, catalytically inactive procaspase 3 was added to control GFP- or Bcr-Abl-expressing Rat-1 lysates in the presence of soluble cytochrome c (1 ng/μl). The processing of caspase 3 was observed via autoradiography. (B) Cell lysates from control GFP- or Bcr-Abl-expressing Rat-1 fibroblasts were incubated with 1 mM dATP and various concentrations of soluble cytochrome c. The processing of endogenous caspase 9 was assayed by Western analysis. Note that the fragment derived from caspase 9-mediated caspase 9 cleavage (the middle of the three cleaved bands indicative that the apoptosome has been activated) is barely visible in the presence of Bcr-Abl. The small amount of (unsuppressed) residual caspase 9 activity in this experiment most likely led to caspase 3 activation, resulting in amplification of caspase 9 processing through caspase 3-mediated caspase 9 cleavage (the upper band of the triplet).

Article Snippet: Human procaspase 9 was immunoprecipitated from human embryonic kidney 293T cells with polyclonal caspase 9 antiserum (2 μg of antibody per mg of total protein; BD Biosciences Pharmingen).

Techniques: In Vitro, Expressing, Autoradiography, Incubation, Western Blot, Derivative Assay, Activity Assay, Activation Assay, Amplification

Journal:

Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

doi: 10.1128/MCB.24.23.10289-10299.2004

Figure Lengend Snippet: Bcr-Abl post-cytochrome c protection in cell lysates occurs by a posttranslational mechanism. (A) Control GFP-expressing cell lysates were preincubated with Abl immunoprecipitates from control GFP- or Bcr-Abl-expressing Rat-1 cells, after which caspase activity was assessed following the addition of 1 ng of soluble cytochrome c per μl. Caspase 3 activity was measured by assessing the cleavage of the colorimetric DEVD-pNA substrate. The results shown are representative of three independent experiments. (B) Control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 lysates were immunoblotted for the Apaf-1 and caspase 9 proteins. (C) Cytosolic Xenopus egg extracts were pretreated with Mos, WT Bcr-Abl (WT), or kinase-dead Bcr-Abl (K671R). ERK activation was assessed by Western immunoblot analysis with an antibody directed against phospho-ERK. OD405, absorbance reading at 405 nm.

Article Snippet: Human procaspase 9 was immunoprecipitated from human embryonic kidney 293T cells with polyclonal caspase 9 antiserum (2 μg of antibody per mg of total protein; BD Biosciences Pharmingen).

Techniques: Expressing, Activity Assay, Activation Assay, Western Blot

Journal:

Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

doi: 10.1128/MCB.24.23.10289-10299.2004

Figure Lengend Snippet: Bcr-Abl signaling does not induce phosphorylation of caspase 9. (A) Endogenous caspase 9 (cas-9) was immunoprecipitated from 293T cells, and in vitro kinase assays were performed with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing cell lysates supplemented with [γ-32P]ATP under conditions in which Bcr-Abl was autophosphorylated (bottom). Lysates were treated with recombinant active ERK (lanes E) as a control for caspase 9 phosphorylation. IgG, immunoglobulin G. (B) control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 fibroblasts were radiolabeled with orthophosphate. Endogenous caspase 9 was captured with a GST fusion protein containing the CARD of Apaf-1. Caspase 9 bound to the Apaf-1 CARD was then subjected to SDS-PAGE and autoradiography or immunoblot analysis with an antibody directed against rat caspase 9. The values on the left are molecular size markers in kilodaltons.

Article Snippet: Human procaspase 9 was immunoprecipitated from human embryonic kidney 293T cells with polyclonal caspase 9 antiserum (2 μg of antibody per mg of total protein; BD Biosciences Pharmingen).

Techniques: Immunoprecipitation, In Vitro, Expressing, Recombinant, SDS Page, Autoradiography, Western Blot

Journal:

Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

doi: 10.1128/MCB.24.23.10289-10299.2004

Figure Lengend Snippet: Bcr-Abl inhibits caspase 9 recruitment to the apoptosome. GST fusion proteins containing the prodomain of caspase 9 (A), the CARD of Apaf-1 (B), or Apaf-1 (1-543) (G) were incubated with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing lysates in the presence of 1 mM dATP. GST proteins were then rebound to glutathione beads (GSH), and the binding ability of Apaf-1 (A) or caspase 9 (B and G) was examined via immunoblot analysis. (C) GST-Apaf-1 (1-543), GST, or GST-Crk protein was incubated with [γ-32P]ATP in either control GFP (lane C)- or Bcr-Abl (lane B)-expressing cell lysates, and radiolabeling was assessed via autoradiography. (D) Apaf-1 was immunoprecipitated from either control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates, and precipitates were immunoblotted (IB) for Apaf-1 or phosphotyrosine (P-Tyr). Anti-Abl immunoprecipitates (IP) were also immunoblotted for P-Tyr as a positive control for the phosphotyrosine Western analysis. (E and F) Control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates were incubated with cytochrome c-Sepharose and 1 mM dATP. The binding ability of Apaf-1 (E) and caspase 9 (F) was then examined by immunoblot analysis.

Article Snippet: Human procaspase 9 was immunoprecipitated from human embryonic kidney 293T cells with polyclonal caspase 9 antiserum (2 μg of antibody per mg of total protein; BD Biosciences Pharmingen).

Techniques: Incubation, Expressing, Binding Assay, Western Blot, Radioactivity, Autoradiography, Immunoprecipitation, Positive Control

Journal: Biomolecules

Article Title: Orientin Induces G0/G1 Cell Cycle Arrest and Mitochondria Mediated Intrinsic Apoptosis in Human Colorectal Carcinoma HT29 Cells

doi: 10.3390/biom9090418

Figure Lengend Snippet: Effect of orientin on cell cycle regulatory proteins. HT29 cells were treated with orientin (6.25 and 12.5 µM) and irinotecan for 24 h. The immunoblot analysis of cyclins, CDKs and their inhibitors involved in G0/G1 phase were carried out. Orientin reduced the expression of cyclins, pRb and CDKs, in contrast, increased the level of p21 WAF1/CIP1 . β-actin was used as an internal control. Quantitative expression of proteins shown after normalization to β-actin. The data presented are the mean ± SD of results from three independent experiments (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control).

Article Snippet: The primary antibodies anti-Bcl-2 (#15071S), Bax (#2772S), Bcl-xL (#2764S), Bid (#2003S), procaspase-3 (#9662S), cleaved caspase-3 (#9661S), procaspase 9(#9508S), cleaved caspase 9 (#9509S), cytochrome C (#11940S), Smac/DIABLO (#2954S), AIF(#4642S), p21 (#2946S), p53 (#9282S), p-Rb (#9307S) or p-H2AX (#2577S) were procured from Cell Signaling Technology, MA.

Techniques: Western Blot, Expressing, Control