Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Time course of DR6-induced apoptosis. After induction of DR6 expression by the addition of tetracycline (Tet), cells were harvested at various time points as indicated and divided into two portions. Half of the harvested cells were directly lysed and separated by SDS-PAGE, followed by Western blot analysis. The other half of the cells were used for preparation of cytosolic extracts and mitochondria-containing membrane fraction as described under “Experimental Procedures.” A, top panel shows induction of DR6 expression. Second panel shows PARP cleavage. Third, fourth, fifth, and sixth panels show activation of caspase-3, caspase-9, caspase-8, and caspase-7, respectively. The activation of caspases was determined by the formation of active forms of these caspases. This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (bottom panel). B, top panels show release of cytochrome c from mitochondria (right column) to the cytosol (left column). Second panels show translocation of Bax from cytosol (left panel) to mitochondria (right panel). Third panel show tBid formation and translocation to mitochondria. Fourth panel shows the reprobe of the second panels with anti-COX I antibody confirming the mitochondria remained intact during preparation.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Expressing, SDS Page, Western Blot, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Death Receptor 6 Induces Apoptosis Not through Type I or Type II Pathways, but via a Unique Mitochondria-dependent Pathway by Interacting with Bax Protein *

doi: 10.1074/jbc.M112.362038

Figure Lengend Snippet: Overexpression of antiapoptotic protein Bcl-2 and Bcl-xL inhibited DR6-induced apoptosis. Cell lysis and fractionation were performed as described previously. A and B, after 24-h transfection, a significant amount of Bcl-2 and Bcl-xL was detected (lanes 3 and 4, top panel). Because the recombinant Bcl-2 and Bcl-xL were expressed as a FLAG-tagged protein, the recombinant Bcl-2 and Bcl-xL were detected with a slower migration rate than endogenous Bcl-2 and Bcl-xL (compare lanes 3 and 4 with lanes 1 and 2). This membrane was also reprobed with anti-actin antibody to indicate relative loading of samples (fourth panel). Second and third panels show tetracycline (Tet)-induced DR6 expression, respectively; fifth and sixth panels, cytochrome c release from mitochondria; seventh and eighth panels, Bax translocation from cytosol to mitochondria. C, top panel shows Western blot for PARP; second to sixth panels, Western blots for caspases; seventh panel, Western blot for actin to indicate relative loading of lysate samples; eighth and ninth panels, Western blots for Bid; tenth and eleventh panels were stained for mitochondrial protein COX I.

Article Snippet: The plasmid pcDNA4/TO/LacZ-Myc-His, which expresses LacZ protein with a Myc tag after the addition of tetracycline, and the conventional plasmid pcDNA3.1/LacZ-Myc-His, which expresses Myc-tagged LacZ protein, were provided in the vector packages by the vendor (Invitrogen).

Techniques: Over Expression, Lysis, Fractionation, Transfection, Recombinant, Migration, Membrane, Expressing, Translocation Assay, Western Blot, Staining