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1.
Figure 3

Figure 3. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

Z-VAD.FMK inhibits the cleavage of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with etoposide (25 μM) in the presence (lane 3) or absence (lane 2) of Z-VAD.FMK (50 μM) as described in Materials and Methods. CPP32 and Ich-1 were detected by Western blot analysis. (Upper panel) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead; (lower panel) 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead. On induction of apoptosis, intact CPP32 (upper panel) and Ich-1 (lower panel) in control cells (lane 1) were cleaved (lane 2), and these were inhibited by Z-VAD.FMK (lane 3).

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.
2.

Figure 4. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

YVAD.CMK inhibits cleavage of lamins A/B but not of CPP32 and Ich-1. THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3). (A) Cell samples were analyzed by Western blot analysis using antibodies to CPP32 and Ich-1 as described in Materials and Methods. (B) Cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of the indicated concentrations of YVAD.CMK (5–25 μM) (lanes 3–5). The cleavage of intact lamin A/B (66 kD) to a fragment of 46 kD was detected by Western blot analysis using a lamin A/B antibody. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (CHX) (25 μM) and TLCK (100 μM) (lane 2) in the presence of Z-VAD.FMK (50 μM) (lane 3) or YVAD.CMK (25 μM) (lane 4). Cell samples were analyzed by Western blot analysis using an antibody to Mch2α.

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.
3.
Figure 1

Figure 1. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

More than one ICE homologue is cleaved in cells exposed to an apoptotic stimulus. THP.1 cells were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM). (A) The time course of induction of apoptosis was determined by flow cytometry as described in Materials and Methods. (B) The time course of cleavage of the proforms of CPP32 (upper panel) and Ich-1 (lower panel) was determined by Western blot analysis as described in Materials and Methods. The 32-kD pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead in the upper panel. The 48-kD pro–Ich-1 is indicated by the upper arrowhead, and the 12-kD cleavage product is indicated by the lower arrowhead in the lower panel.

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.
4.
Figure 5

Figure 5. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

Z.DEVD-AFC cleavage activity in lysates derived from THP.1 cells committed to apoptosis precedes the morphological characteristics of cell death. THP.1 cells were incubated for up to 4 h, either alone (Con) (○–○) or in the presence of cycloheximide (CHX) and TLCK (•–•; ▪–▪), and lysates were prepared at the indicated time points as described in Materials and Methods. Z-DEVD.AFC (○–○; •–•) and Ac-YVAD.AMC (▪–▪) hydrolysis activities were determined as described in Materials and Methods and are shown in A. The extent of apoptosis at each time point, as assessed by flow cytometry, is indicated in brackets. Lysates from both untreated (Con) and THP.1 cells exposed to an apoptotic stimulus (CHX + TLCK) were analyzed by Western blot analysis using antibodies to CPP32 as described in Materials and Methods. (B) pro-CPP32 is indicated by the upper arrowhead, and the 17-kD cleavage product is indicated by the lower arrowhead. The time course of cleavage of CPP32 (B) paralleled the hydrolysis activity of the lysates towards Z-DEVD.AFC.

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.
5.
Figure 6

Figure 6. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

Processing of Mch3α in apoptotic THP.1 cells: inhibition by ZVAD.FMK but not by YVAD.CMK. (A) THP.1 cells were incubated for up to 4 h, either alone (Con) or in the presence of cycloheximide (CHX) (25 μM) and TLCK (100 μM). Where indicated, cells were pretreated for 1 h with Z-VAD.FMK (50 μM), and then incubated for 4 h in the presence of the apoptotic stimulus. The time course of processing of the proform of Mch3α (lanes 1–5) and its inhibition by Z-VAD.FMK (lane 6) was determined by Western blot analysis as described in Materials and Methods. (B) THP.1 cells were incubated for 4 h in the presence of etoposide (25 μM), stained with Hoechst 33342 and propidium iodide, and then sorted by flow cytometry as previously described (see Materials and Methods and Fig. ). Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis. (C) THP.1 cells were incubated for 4 h, either alone (lane 1) or with cycloheximide (25 μM) and TLCK (100 μM) (lane 2) in the presence of YVAD.CMK (25 μM) (lane 3), and samples were analyzed by Western blot analysis. The proforms of Mch3α and its cleavage product are indicated by the upper and lower arrowheads, respectively (A–C).

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.
6.

Figure 2. From: Processing/Activation of At Least Four Interleukin-1β Converting Enzyme–like Proteases Occurs during the Execution Phase of Apoptosis in Human Monocytic Tumor Cells.

CPP32 and Ich-1 are extensively cleaved in apoptotic but not morphologically normal THP.1 cells, and their processing is concomitant with the cleavage of PARP, U1-70K, and lamins A/ B. THP.1 cells were incubated for 4 h in the presence of etoposide (25 μM), stained with Hoechst 33342 and propidium iodide, and then sorted by flow cytometry as described previously (). Cells with low blue fluorescence were morphologically normal and those with high blue fluorescence exhibited distinctive apoptotic morphology when examined by fluorescence microscopy. Cells with either normal (lane 1) or apoptotic (lane 2) morphology were analyzed by Western blot analysis as described in Materials and Methods. (A) Cells were analyzed using antibodies to CPP32 (upper panel) and Ich-1 (lower panel). (B) Cells were analyzed using antibodies to PARP, U1-70K, and lamins A/B (upper, middle and lower panels, respectively). The proforms of CPP32 and Ich-1 are indicated by the upper arrowheads (A), and intact PARP, U170K, and lamins A/B are indicated by the upper arrowheads (B). The lower arrowheads represent either processed enzymes or cleaved proteolytic fragments. Cells displaying normal morphology contain primarily the intact forms of all the proteins analyzed (lane 1), whereas, in apoptotic cells, processing of more than one ICE-like protease is detected and associated with cleavage of PARP, U1-70K, and lamins A/B.

Marion MacFarlane, et al. J Cell Biol. 1997 April 21;137(2):469-479.

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