Figure 1.
Figure 1. Processing of procaspases-2 and -8 in total cellular lysates on CD95 stimulation. (A) B-lymphoblastoid SKW6.4 cells were treated with 5 μg/mL agonistic anti–APO-1 antibody for indicated periods of time. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (B) Two cleavage steps of procaspase-2 during apoptosis. At the first cleavage step p33 and small catalytical subunit (p12) are formed. As a result of the second cleavage step the CARD-containing prodomain is generated along with the large catalytical subunit. The epitope of anti–caspase-2 mAbs is shown by black arrow. (C) B-lymphoblastoid SKW6.4 cells were treated with 200 ng/mL agonistic anti–APO-1 antibody for indicated periods of time. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (D) CEM cells were treated with 5 μg/mL agonistic anti-CD95 (anti–APO-1) antibody for indicated time points. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (E) H9 cells were treated with 1 μg/mL agonistic anti-CD95 (anti–APO-1) antibody for indicated time points. (F) SKW6.4 cells were treated with 5, 10, and 20 μg/mL agonistic anti–APO-1 antibody for 1 hour. The activities of caspase-2 and -8 in cellular lysates were measured using zVDVAD-afc and zIETD-afc, correspondingly. (G) SKW6.4 cells were pretreated with indicated concentrations of zVAD-fmk for 30 minutes. Subsequently, SKW6.4 cells were stimulated with 1 μg/mL agonistic anti–APO-1 antibody for 1 hour. The activity of caspase-2 and -8 in cellular lysates was measured using zVDVAD-afc and zIETD-afc, respectively. (F-G) Error bars indicate SD. (H) SKW6.4 cells were treated with 1 μg/mL LZ-CD95L for indicated time points. ZVAD-fmk was used, when indicated, in a concentration of 50 μM. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8).

Processing of procaspases-2 and -8 in total cellular lysates on CD95 stimulation. (A) B-lymphoblastoid SKW6.4 cells were treated with 5 μg/mL agonistic anti–APO-1 antibody for indicated periods of time. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (B) Two cleavage steps of procaspase-2 during apoptosis. At the first cleavage step p33 and small catalytical subunit (p12) are formed. As a result of the second cleavage step the CARD-containing prodomain is generated along with the large catalytical subunit. The epitope of anti–caspase-2 mAbs is shown by black arrow. (C) B-lymphoblastoid SKW6.4 cells were treated with 200 ng/mL agonistic anti–APO-1 antibody for indicated periods of time. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (D) CEM cells were treated with 5 μg/mL agonistic anti-CD95 (anti–APO-1) antibody for indicated time points. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). (E) H9 cells were treated with 1 μg/mL agonistic anti-CD95 (anti–APO-1) antibody for indicated time points. (F) SKW6.4 cells were treated with 5, 10, and 20 μg/mL agonistic anti–APO-1 antibody for 1 hour. The activities of caspase-2 and -8 in cellular lysates were measured using zVDVAD-afc and zIETD-afc, correspondingly. (G) SKW6.4 cells were pretreated with indicated concentrations of zVAD-fmk for 30 minutes. Subsequently, SKW6.4 cells were stimulated with 1 μg/mL agonistic anti–APO-1 antibody for 1 hour. The activity of caspase-2 and -8 in cellular lysates was measured using zVDVAD-afc and zIETD-afc, respectively. (F-G) Error bars indicate SD. (H) SKW6.4 cells were treated with 1 μg/mL LZ-CD95L for indicated time points. ZVAD-fmk was used, when indicated, in a concentration of 50 μM. The cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8).

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