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Results: 7

1.
Figure 7

Figure 7. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

Schematic diagram of the proposed model. Cain/cabin1 is cleaved by calpain to be inactivated, and Cn is subsequently activated to mediate calcium-ionophore-induced cell death.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
2.
Figure 6

Figure 6. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

In vitro binding assay identifying the cain/cabin1-binding domain in the Cn A subunit. (A) Schematic diagram showing deletion mutants of Cn A (CnA): The CnAΔCaMAI mutant lacks the calmodulin (CaM)-binding and autoinhibitory (AI) domains and the CnAΔeD mutant lacks Cn B (CnB)-binding region and contains only the catalytic domain. (B) Purified GST and GST-cain/cabin1-C were subjected to in vitro binding assays by using [35S]methionine-labeled full-length or deletions of Cn A. Interacting proteins bound to GST-cain/cabin1-C were pulled down with GST-Sepharose, separated by SDS/PAGE, and exposed to x-ray film for autoradiography.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
3.
Figure 1

Figure 1. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

In vitro cleavage of cain/cabin1 by purified m-calpain. (A) Small pools of cDNA (approximately 100) were translated in vitro in the presence of [35S]methionine, and then incubated with m-calpain (0.165 unit/ml). The reaction products and untreated controls were separated by SDS/PAGE and then exposed to x-ray film. Arrows indicate Cain/cabin1 and its cleavage product. (B) The C terminus of mouse cain/cabin1 (cain/cabin1-C) was labeled with [35S]methionine and incubated with the indicated amounts of m-calpain at 30°C for 1 h. ΔCain/cabin1-C indicates a cleavage product of cain/cabin1-C. (C) Cain/cabin1 was transiently expressed in HEK293T cells. One day later, cell extracts were prepared and incubated with m-calpain (0.75 unit/ml) in the presence or absence of calpeptin (20 μg/ml). The reaction products were analyzed by Western blotting with anti-cain/cabin1 antibody.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
4.
Figure 4

Figure 4. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

Attenuation of A23187-induced cell death by treatment with FK506 and expression of either cain/cabin1, an inactive Cn mutant [CnAβ2(1–401/H160N)], or Cn deletions. (A) The effects of FK506 on A23187-induced cell death. Jurkat cells were exposed to A23187 (1 μM) for 4 h in the presence of the indicated amounts of FK506, and cell viability (percent of cell death) was determined. (B) Jurkat cells were transiently cotransfected with pEGFP-N1 and either pcDNA3, pCnAβ2(1–401/H160N), or pCain/cabin1. One day later, cells were exposed to A23187 for the indicated times, and cell viability was determined on the basis of the morphology of GFP-positive cells under a fluorescence microscope. (C) Effect of cain/cabin1 deletions, cain/cabin1-C and cain/cabin1-CΔa (see diagram in Fig. 5A), on A23187-induced cell death. Jurkat cells were transfected for 24 h with pEGFP-N1 and either pcDNA3-HA, pHA-cain/cabin1-C, or pHA-cain/cabin1-CΔa and then exposed to A23187 for the indicated times. The data shown are the means ± SD from at least three independent experiments.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
5.
Figure 3

Figure 3. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

Suppression of A23187-induced Cn activation by calpain inhibitors in vitro and in vivo. (A) Activity assay of calpain in vivo. Jurkat cells were exposed to A23187 (1 μM) for the indicated times, and calpain activation was then examined by the cell-permeable fluorogenic calpain substrate (Rhodamine-110). (B) Activity assay of Cn in vitro. Cell extracts prepared from Jurkat cells exposed to A23187 were assayed for Cn activity. The Cn activity determined from four independent experiments is indicated as picomoles of phosphate per minute per microgram of protein. (C) Calpain-mediated activation of Cn. Jurkat cells were exposed for 4 h to A23187 in the presence or absence of calpeptin (40 μg/ml) or zLLY (20 μM), and cell extracts were then assayed for the Cn activity. (D) Calpain-mediated activation of Cn in vivo. Jurkat cells were transfected with pNF-AT-luc and pβactgal as an internal control. After 48 h, cells were treated with A23187 for 4 h in the presence or absence of calpeptin (40 μg/ml), zLLY (20 μM), or FK506 (3 μM), and luciferase activity was then measured.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
6.
Figure 5

Figure 5. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

In vitro mapping of calpain-sensitive region in the C terminus of cain/cabin1 and intracellular interaction with Cn A. (A) Schematic diagram of cain/cabin1 and its deletions, cain/cabin1-C, cain/cabin1-CΔa, and cain/cabin1-CΔb. Gray box indicates the Cn-binding domain (CBD). (B) Lack of calpain-mediated cleavage in the cain/cabin1-CΔa. Cain/cabin1-C and cain/cabin1-CΔa were labeled with [35S]methionine and subjected to in vitro cleavage assays. Asterisk indicates a cleavage product of cain/cabin1-C. (C) Purified GST, GST-cain/cabin1-C, and GST-cain/cabin1-CΔb fusion proteins were incubated with calpain (0.005 unit) at 30°C for the indicated times and then visualized by Coomassie blue staining after SDS/PAGE. Asterisks indicate the cleavage product of respective protein. (D) Intracellular binding assays. HEK293 cells were transiently cotransfected with pHA-CnA, pHA-CnB, and either pHA-cain/cabin1-C or pHA-cain/cabin1-CΔa. One day later, proteins of HEK293 cell extracts were immunoprecipitated with preimmune serum (Pre) or anti-cain/cabin1 antibody (IP), and immunoblotted with anti-Cn A antibody. Cn A and heavy chain (H.C.) of Ig are indicated with arrows in the immunoprecipitates (Middle) and in total cell extracts (Left).

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.
7.
Figure 2

Figure 2. From: Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

Calpain-dependent cleavage of cain/cabin1 during A23187-induced cell death. (A) Jurkat cells were incubated with A23187 (1 μM) for the indicated times and cell viability (percent of cell death) was determined by trypan blue exclusion. Bars represent means ± SD from at least four independent experiments. (B) Cell extracts were analyzed by Western blotting with anti-cain/cabin1 antibody. Cain/cabin1 and Δcain/cabin1 indicate full-length (230 kDa) and a cleavage product (32 kDa) of cain/cabin1, respectively. (C) Jurkat cells were exposed to A23187 (1 μM) for 3 h in the presence or absence of the calpain inhibitors, calpeptin (40 μg/ml) and zLLY (20 μM), and analyzed by Western blotting. (D) Internucleosomal DNA fragmentation assays. Jurkat cells were exposed to A23187, and tumor necrosis factor-related apoptosis-inducing ligand (100 ng/ml) was used as a positive control of apoptosis. (E) Proteolytic activation of caspase-3 during A23187-induced cell death was assessed by Western blot analysis. P18 indicates the processed subunit (18 kDa) of caspase-3.

Min-Jung Kim, et al. Proc Natl Acad Sci U S A. 2002 July 23;99(15):9870-9875.

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