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adam10  (R&D Systems)
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FIGURE 3. Anx A1 cleavage is downstream of <t>ADAM10.</t> A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.
Adam10, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant human adam10  (R&D Systems)
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R&D Systems recombinant human adam10
FIGURE 3. Anx A1 cleavage is downstream of <t>ADAM10.</t> A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.
Recombinant Human Adam10, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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FIGURE 3. Anx A1 cleavage is downstream of ADAM10. A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 3. Anx A1 cleavage is downstream of ADAM10. A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Inhibition, Western Blot, Membrane, Recombinant, Incubation, Control, Knockdown, Quantitative RT-PCR, Expressing, Electroporation

FIGURE 4. Anx A1 is directly cleaved by ADAM10 after F7. A, Domain structure of different anx A1 constructs. Arabic numbers depict the amino acid position, and annexin repeats are numbered I–IV. B, Recombinant human anx A1 (aa 1–346) is processed by recombinant human ADAM10. One mi- crogram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng recombinant human ADAM10 ectodomain in the presence or absence of 100 mM of the matrix metalloproteinase inhibitor TAPI-2 at 37˚C for the indicated times. Subsequently, anx A1 cleavage was detected by SDS-PAGE and immunoblot analysis with an anti-anx A1 Ab. C, Recombinant human anx A1 core domain (aa 47–346) is not cleaved by ADAM10. Incubation of anx A1 (aa 47–346) with ADAM10 ectodomain was performed as in B. For immunoblot analysis, a polyclonal anti-anx A1 Ab was used. D, The cleavage site of ADAM10 is located within the unique N-terminal domain of anx A1 (aa 1–46). One microgram of the recombinant human anx A1 N-terminal domain (aa 1–46) was incubated with ADAM10 as in B. Cleavage fragments were separated by SDS-PAGE and visualized by subsequent silver staining. E, Recombinant human anx A1 (aa 1–346) is not processed by recombinant human ADAM17. One microgram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng of recombinant human ADAM17 ectodomain as in B. F, Identification of the ADAM10 cleavage site within the anx A1 N-terminal domain. Recombinant human anx A1 (aa 1–346) was incubated with native or heat-inactivated recombinant human ADAM10 as in A. Subsequently, the reaction mixture was subjected to N-terminal Edman degradation. The N-terminal sequence newly generated by incubation with active ADAM10 was L8

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 4. Anx A1 is directly cleaved by ADAM10 after F7. A, Domain structure of different anx A1 constructs. Arabic numbers depict the amino acid position, and annexin repeats are numbered I–IV. B, Recombinant human anx A1 (aa 1–346) is processed by recombinant human ADAM10. One mi- crogram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng recombinant human ADAM10 ectodomain in the presence or absence of 100 mM of the matrix metalloproteinase inhibitor TAPI-2 at 37˚C for the indicated times. Subsequently, anx A1 cleavage was detected by SDS-PAGE and immunoblot analysis with an anti-anx A1 Ab. C, Recombinant human anx A1 core domain (aa 47–346) is not cleaved by ADAM10. Incubation of anx A1 (aa 47–346) with ADAM10 ectodomain was performed as in B. For immunoblot analysis, a polyclonal anti-anx A1 Ab was used. D, The cleavage site of ADAM10 is located within the unique N-terminal domain of anx A1 (aa 1–46). One microgram of the recombinant human anx A1 N-terminal domain (aa 1–46) was incubated with ADAM10 as in B. Cleavage fragments were separated by SDS-PAGE and visualized by subsequent silver staining. E, Recombinant human anx A1 (aa 1–346) is not processed by recombinant human ADAM17. One microgram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng of recombinant human ADAM17 ectodomain as in B. F, Identification of the ADAM10 cleavage site within the anx A1 N-terminal domain. Recombinant human anx A1 (aa 1–346) was incubated with native or heat-inactivated recombinant human ADAM10 as in A. Subsequently, the reaction mixture was subjected to N-terminal Edman degradation. The N-terminal sequence newly generated by incubation with active ADAM10 was L8

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Construct, Recombinant, Incubation, SDS Page, Western Blot, Silver Staining, Sequencing, Generated

FIGURE 6. ADAM10 contributes to the release of “find-me” signals from secondary necrotic cells. A, GM 6001 inhibits the release of monocytic chemoattractants during secondary necrosis. Jurkat, MOLT-4, and THP-1 cells were UV-irradiated and incubated for 12 or 18 h in the presence or absence of the broad-range matrix metalloproteinase inhibitor GM 6001 (100 mM). Cell-free culture supernatants were collected and analyzed for their chemotactic potential as in Fig. 5A. Error bars represent SD of quadruplicates. B, The release of secondary necrotic cell-derived attraction signals is strongly decreased in the presence of the ADAM10-specific inhibitor GI 254023X. Jurkat cells were UV-irradiated and incubated for 18 h in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Supernatants were collected and applied to a transmigration assay with THP-1 cells as in Fig. 5A. Error bars represent SD of quadruplicates. C, Evaluation of ADAM10 knockdown efficiency by FACS analysis. Knockdown of ADAM10 expression in Jurkat cells was carried out as in Fig. 3G. On day 5 after the first electroporation, cells were fixed, permeabilized, and stained with anti-ADAM10–PE Ab to detect ADAM10 expression level by FACS analysis or IgG-2b-PE isotype control. Left panel, Representative histograms are shown. Right panel, Median PE fluorescence of the histograms in the left panel. D, The release of monocytic attraction signals is strongly decreased in ADAM10-silenced secondary necrotic cells. ADAM10-silenced cells were induced to undergo secondary necrosis by UV irradiation. Cell culture supernatants were harvested and analyzed for their chemotactic potential in a transmigration assay with THP-1 cells as in Fig. 5A. Mean values + SD of quadruplicates are given.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 6. ADAM10 contributes to the release of “find-me” signals from secondary necrotic cells. A, GM 6001 inhibits the release of monocytic chemoattractants during secondary necrosis. Jurkat, MOLT-4, and THP-1 cells were UV-irradiated and incubated for 12 or 18 h in the presence or absence of the broad-range matrix metalloproteinase inhibitor GM 6001 (100 mM). Cell-free culture supernatants were collected and analyzed for their chemotactic potential as in Fig. 5A. Error bars represent SD of quadruplicates. B, The release of secondary necrotic cell-derived attraction signals is strongly decreased in the presence of the ADAM10-specific inhibitor GI 254023X. Jurkat cells were UV-irradiated and incubated for 18 h in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Supernatants were collected and applied to a transmigration assay with THP-1 cells as in Fig. 5A. Error bars represent SD of quadruplicates. C, Evaluation of ADAM10 knockdown efficiency by FACS analysis. Knockdown of ADAM10 expression in Jurkat cells was carried out as in Fig. 3G. On day 5 after the first electroporation, cells were fixed, permeabilized, and stained with anti-ADAM10–PE Ab to detect ADAM10 expression level by FACS analysis or IgG-2b-PE isotype control. Left panel, Representative histograms are shown. Right panel, Median PE fluorescence of the histograms in the left panel. D, The release of monocytic attraction signals is strongly decreased in ADAM10-silenced secondary necrotic cells. ADAM10-silenced cells were induced to undergo secondary necrosis by UV irradiation. Cell culture supernatants were harvested and analyzed for their chemotactic potential in a transmigration assay with THP-1 cells as in Fig. 5A. Mean values + SD of quadruplicates are given.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Irradiation, Incubation, Derivative Assay, Transmigration Assay, Knockdown, Expressing, Electroporation, Staining, Control, Cell Culture