fluorogenic substrates for measuring activity of adam10 and adam17 Search Results


anti adam17 antibody  (Bioss)
92
Bioss anti adam17 antibody
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Anti Adam17 Antibody, supplied by Bioss, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp adam17 mm00456428 m1  (Thermo Fisher)
94
Thermo Fisher gene exp adam17 mm00456428 m1
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Gene Exp Adam17 Mm00456428 M1, supplied by Thermo Fisher, 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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gene exp ctnnb1 hs00355049 m1  (Thermo Fisher)
99
Thermo Fisher gene exp ctnnb1 hs00355049 m1
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Gene Exp Ctnnb1 Hs00355049 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mocac lys pro leu gly leu dap dnp ala arg nh 2  (Biosynth Carbosynth)
95
Biosynth Carbosynth mocac lys pro leu gly leu dap dnp ala arg nh 2
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Mocac Lys Pro Leu Gly Leu Dap Dnp Ala Arg Nh 2, supplied by Biosynth Carbosynth, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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incb8765  (Incyte corporation)
90
Incyte corporation incb8765
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Incb8765, supplied by Incyte corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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adam10/adam17 small molecule inhibitor aderbasib  (Incyte corporation)
90
Incyte corporation adam10/adam17 small molecule inhibitor aderbasib
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Adam10/Adam17 Small Molecule Inhibitor Aderbasib, supplied by Incyte corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp tnfsf11 mm01313944 g1  (Thermo Fisher)
86
Thermo Fisher gene exp tnfsf11 mm01313944 g1
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Gene Exp Tnfsf11 Mm01313944 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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adam10 adam17 inhibitor tapi 2  (Biosynth Carbosynth)
90
Biosynth Carbosynth adam10 adam17 inhibitor tapi 2
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
Adam10 Adam17 Inhibitor Tapi 2, supplied by Biosynth Carbosynth, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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adam17 crispr cas9 knock out  (Addgene inc)
96
Addgene inc adam17 crispr cas9 knock out
ADAM10 and <t>ADAM17</t> are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.
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adam17 sirna  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology adam17 sirna
PrAMA approach for solid tissue analysis. ( A ) Summary table and heat-map show 7 FRET-based polypeptide substrates with cleavage sequences (HomoPhe, homophenylalanine; Cha, 3-cyclohexylalanine), endogenous-protein protease substrates from which the peptides were designed, and the catalytic efficiencies with which various recombinant proteases cleave the substrates (for more information see ref. 35). ( B ) Workflow illustrates PrAMA for cell and tissue lysates. ( C ) Proof-of-principle is shown for PrAMA applied to known solutions, and one combination, of recombinant enzymes (abbreviations: rM2, recombinant MMP2; rA10, recombinant ADAM10; rA17, recombinant <t>ADAM17).</t> Normalized cleavage rates for each of the 4 solutions were measured across the 7 substrates (left), and data were interpreted using known enzyme-substrate preferences [as in ( A )] to infer which recombinant enzymes were present in the mixture. Actual mixture composition (top right) and PrAMA results (bottom right) are presented. ( D ) Surface plots depict three-dimensional “systematic PrAMA” inference as a function of the two parameters sensitivity (Syntherror) and specificity (Sigmathreshold). Processing data of seven substrates obtained with recombinant MMP2, ADAM10, and ADAM17 were analyzed by PrAMA across varying combinations of Syntherror and Sigmathreshold parameters to reveal how these two parameters influence PrAMA sensitivity and specificity. The three rows of surface-plots correspond to the analyzed individual three recombinant enzyme solutions (rMMP2, rADAM10 and rADAM17), and the three columns of surface-plots correspond to the three individual PrAMA-inferred enzyme activities from these solutions (MMP2a, ADAM10sa and ADAM17sa). The color scale ranges from red to blue, which reflects the surface heights as labeled on the vertical axis. The three-dimensional surface plots shown in the figure depict a representation of the two-dimensional “systematic PrAMA” shown in Figs. C- H. In the latter cases (Figs. C-H) and the rest of presented data, Syntherror is held constant (0.5) across a range of Sigmathreshold values. The experimental details follow those described for Fig. C.
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adam10 inhibition assays proteolytic activities  (R&D Systems)
94
R&D Systems adam10 inhibition assays proteolytic activities
RTDs inhibit cellular TACE and <t>ADAM10</t> sheddase activities. A and B, RTDs, but not acyclic S7 or α-defensin HNP-4, suppress TACE-mediated ectodomain shedding of TGFα in COS7 cells. Data are expressed as fold-TGFα shedding relative to constitutive TGFα release. C, RTD-1, but not S7 or HNP-4, inhibits ADAM10-dependent cleavage of BTC in COS7 cells. Data are expressed as fold BTC shedding relative to constitutive BTC release. Data in panels A--C represent mean ± S.D. of a representative experiment containing 3 technical repeats. D, RTDs inhibit rADAM10 proteolysis of its fluorogenic substrate. Enzyme reactions were performed for 30 min at 37 °C with 1 nm ADAM10 and 10 μm substrate (R&D Systems, ES010). Enzyme inhibition is expressed as percent change in substrate conversion rate relative to peptide-free controls. Data represent mean ± S.D. of 2 independent experiments containing 2 technical repeats each.
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mmps preclinical ra wyeth  (Biosynth Carbosynth)
90
Biosynth Carbosynth mmps preclinical ra wyeth
RTDs inhibit cellular TACE and <t>ADAM10</t> sheddase activities. A and B, RTDs, but not acyclic S7 or α-defensin HNP-4, suppress TACE-mediated ectodomain shedding of TGFα in COS7 cells. Data are expressed as fold-TGFα shedding relative to constitutive TGFα release. C, RTD-1, but not S7 or HNP-4, inhibits ADAM10-dependent cleavage of BTC in COS7 cells. Data are expressed as fold BTC shedding relative to constitutive BTC release. Data in panels A--C represent mean ± S.D. of a representative experiment containing 3 technical repeats. D, RTDs inhibit rADAM10 proteolysis of its fluorogenic substrate. Enzyme reactions were performed for 30 min at 37 °C with 1 nm ADAM10 and 10 μm substrate (R&D Systems, ES010). Enzyme inhibition is expressed as percent change in substrate conversion rate relative to peptide-free controls. Data represent mean ± S.D. of 2 independent experiments containing 2 technical repeats each.
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Image Search Results


ADAM10 and ADAM17 are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: ADAM10 and ADAM17 are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Flow Cytometry, Staining, Transfection, shRNA, Western Blot

Inhibition of ADAM10 and ADAM17 increases Robo4 C-terminal Fragment, and Robo4 co-localizes with ADAM10 and ADAM17 in endothelial cells. ( A ) Human Robo4-HA-FLAG expression. The expression of hRobo4-HA-FLAG in dEC lysate was probed with an anti-human Robo4 ectodomain antibody in Western blot. A 130 KDa band was detected. ( B ) Pharmacological inhibition of ADAM10 and ADAM17 decreased Robo4-CTF. hRobo4-HA-FLAG transiently expressing dECs were treated with vehicle control DMSO or GW (6 μM) with or without Mg132 (12 μM) or chloroquine diphosphate (CD, 50 μM). The cell lysates were probed with an anti-FLAG antibody. The hRobo4 CTF was normalized to β-actin and further normalized to the control group. GW and Mg132 decreased hRobo4-CTF. ( C ) Knockdown of ADAM10 or ADAM17 decreased Robo4 CTF. The dECs were co-transected with hRobo4-HA-FLAG and scrambled or ADAM knockdown constructs and then probed with an anti-FLAG antibody in western blot. ( D ) Inhibition of ADAM10 and ADAM17 decreased endogenous Robo4-CTF. Vehicle DMSO or GW (6 μM)-treated dECs were lysed and probed with an anti-mouse Robo4 CTF antibody in western blot. No Robo4 bands were detected in the Robo4 KO dEC cell lysate. ( E ) Robo4 co-localizes with ADAM10 and ADAM17. dECs were transiently expressed with hRobo4-HA-FLAG and stained for HA and endogenous ADAM10 or ADAM17 with corresponding antibodies. The merge yellow fluorescence shows Robo4-HA (green) co-localized with ADAM10 or ADAM17 (red). Robo4-HA-FLAG co-localized with ADAM10 or ADAM17 with overlap coefficients of 0.900 and 0.834, respectively. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. *p < 0.05; **p < 0.01.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Inhibition of ADAM10 and ADAM17 increases Robo4 C-terminal Fragment, and Robo4 co-localizes with ADAM10 and ADAM17 in endothelial cells. ( A ) Human Robo4-HA-FLAG expression. The expression of hRobo4-HA-FLAG in dEC lysate was probed with an anti-human Robo4 ectodomain antibody in Western blot. A 130 KDa band was detected. ( B ) Pharmacological inhibition of ADAM10 and ADAM17 decreased Robo4-CTF. hRobo4-HA-FLAG transiently expressing dECs were treated with vehicle control DMSO or GW (6 μM) with or without Mg132 (12 μM) or chloroquine diphosphate (CD, 50 μM). The cell lysates were probed with an anti-FLAG antibody. The hRobo4 CTF was normalized to β-actin and further normalized to the control group. GW and Mg132 decreased hRobo4-CTF. ( C ) Knockdown of ADAM10 or ADAM17 decreased Robo4 CTF. The dECs were co-transected with hRobo4-HA-FLAG and scrambled or ADAM knockdown constructs and then probed with an anti-FLAG antibody in western blot. ( D ) Inhibition of ADAM10 and ADAM17 decreased endogenous Robo4-CTF. Vehicle DMSO or GW (6 μM)-treated dECs were lysed and probed with an anti-mouse Robo4 CTF antibody in western blot. No Robo4 bands were detected in the Robo4 KO dEC cell lysate. ( E ) Robo4 co-localizes with ADAM10 and ADAM17. dECs were transiently expressed with hRobo4-HA-FLAG and stained for HA and endogenous ADAM10 or ADAM17 with corresponding antibodies. The merge yellow fluorescence shows Robo4-HA (green) co-localized with ADAM10 or ADAM17 (red). Robo4-HA-FLAG co-localized with ADAM10 or ADAM17 with overlap coefficients of 0.900 and 0.834, respectively. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Inhibition, Expressing, Western Blot, Construct, Staining, Fluorescence

sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Generated

PrAMA approach for solid tissue analysis. ( A ) Summary table and heat-map show 7 FRET-based polypeptide substrates with cleavage sequences (HomoPhe, homophenylalanine; Cha, 3-cyclohexylalanine), endogenous-protein protease substrates from which the peptides were designed, and the catalytic efficiencies with which various recombinant proteases cleave the substrates (for more information see ref. 35). ( B ) Workflow illustrates PrAMA for cell and tissue lysates. ( C ) Proof-of-principle is shown for PrAMA applied to known solutions, and one combination, of recombinant enzymes (abbreviations: rM2, recombinant MMP2; rA10, recombinant ADAM10; rA17, recombinant ADAM17). Normalized cleavage rates for each of the 4 solutions were measured across the 7 substrates (left), and data were interpreted using known enzyme-substrate preferences [as in ( A )] to infer which recombinant enzymes were present in the mixture. Actual mixture composition (top right) and PrAMA results (bottom right) are presented. ( D ) Surface plots depict three-dimensional “systematic PrAMA” inference as a function of the two parameters sensitivity (Syntherror) and specificity (Sigmathreshold). Processing data of seven substrates obtained with recombinant MMP2, ADAM10, and ADAM17 were analyzed by PrAMA across varying combinations of Syntherror and Sigmathreshold parameters to reveal how these two parameters influence PrAMA sensitivity and specificity. The three rows of surface-plots correspond to the analyzed individual three recombinant enzyme solutions (rMMP2, rADAM10 and rADAM17), and the three columns of surface-plots correspond to the three individual PrAMA-inferred enzyme activities from these solutions (MMP2a, ADAM10sa and ADAM17sa). The color scale ranges from red to blue, which reflects the surface heights as labeled on the vertical axis. The three-dimensional surface plots shown in the figure depict a representation of the two-dimensional “systematic PrAMA” shown in Figs. C- H. In the latter cases (Figs. C-H) and the rest of presented data, Syntherror is held constant (0.5) across a range of Sigmathreshold values. The experimental details follow those described for Fig. C.

Journal: Journal of Cancer

Article Title: Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

doi: 10.7150/jca.20779

Figure Lengend Snippet: PrAMA approach for solid tissue analysis. ( A ) Summary table and heat-map show 7 FRET-based polypeptide substrates with cleavage sequences (HomoPhe, homophenylalanine; Cha, 3-cyclohexylalanine), endogenous-protein protease substrates from which the peptides were designed, and the catalytic efficiencies with which various recombinant proteases cleave the substrates (for more information see ref. 35). ( B ) Workflow illustrates PrAMA for cell and tissue lysates. ( C ) Proof-of-principle is shown for PrAMA applied to known solutions, and one combination, of recombinant enzymes (abbreviations: rM2, recombinant MMP2; rA10, recombinant ADAM10; rA17, recombinant ADAM17). Normalized cleavage rates for each of the 4 solutions were measured across the 7 substrates (left), and data were interpreted using known enzyme-substrate preferences [as in ( A )] to infer which recombinant enzymes were present in the mixture. Actual mixture composition (top right) and PrAMA results (bottom right) are presented. ( D ) Surface plots depict three-dimensional “systematic PrAMA” inference as a function of the two parameters sensitivity (Syntherror) and specificity (Sigmathreshold). Processing data of seven substrates obtained with recombinant MMP2, ADAM10, and ADAM17 were analyzed by PrAMA across varying combinations of Syntherror and Sigmathreshold parameters to reveal how these two parameters influence PrAMA sensitivity and specificity. The three rows of surface-plots correspond to the analyzed individual three recombinant enzyme solutions (rMMP2, rADAM10 and rADAM17), and the three columns of surface-plots correspond to the three individual PrAMA-inferred enzyme activities from these solutions (MMP2a, ADAM10sa and ADAM17sa). The color scale ranges from red to blue, which reflects the surface heights as labeled on the vertical axis. The three-dimensional surface plots shown in the figure depict a representation of the two-dimensional “systematic PrAMA” shown in Figs. C- H. In the latter cases (Figs. C-H) and the rest of presented data, Syntherror is held constant (0.5) across a range of Sigmathreshold values. The experimental details follow those described for Fig. C.

Article Snippet: Human ADAM10 and ADAM17 siRNA (pools of three target specific siRNA), control (scrambled siRNA), siRNA transfection medium, and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Recombinant, Labeling

Modified PrAMA detects ADAM10sa and ADAM17sa presence in wild-type and absence in ADAM10 -/- and ADAM17 -/- MEF lysates, respectively. ADAM10 +/- , ADAM10 -/- ( A, C, E ) , ADAM17 +/+ and ADAM17 -/- ( B, D, F ) MEFs were activated with PMA/Ionomycin and lysed. The 150 μL Tris solutions of 2 μg cell lysates, 10 μM PEPDAB substrates and 0.5% Roche/Halt protease inhibitors were incubated for 4 h at 37 o C, and the developed fluorescence was recorded hourly using TECAN fluorimeter. The presented experiments are representative of 5 performed. The enzymatic activity data are shown as pM means of duplicate measurements ± SD of the processed substrates ( A, B ). The decreased processing of PEPDAB005, 010, 011, 014 and 022 with knockout MEF lysates is significant ( ADAM10 +/- vs ADAM10 -/- MEFs and ADAM17 +/+ vs ADAM17 -/- MEFs: p<0.0001 and p<0.001, p=0.0056 and p<0.001, p=0.0016 and p=0.0025, p=0.0016 and p=0.009, and p=0.006 and p=0.004, respectively). Systematic PrAMA data using Syntherror/Sigmathreshold parameters 0.5/0.0 to 0.5/2.0 and 0.5/0.0 to 0.5/1.0 are presented as ADAM10sa AU ( C ) and ADAM17sa AU ( D ) , respectively. Standard errors of PrAMA data were 2.5% to 5.8%. True-positive ADAM10sa and ADAM17sa in ADAM10 +/- and ADAM17 +/+ MEFs, respectively, are presented as % of specific enzyme activities in wild-type MEFs, containing both the true-positive and false-positive activities, relative to ADAM10 -/- (E) and ADAM17 -/- (F) ME Fs containing only the false-positive activities, respectively.

Journal: Journal of Cancer

Article Title: Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

doi: 10.7150/jca.20779

Figure Lengend Snippet: Modified PrAMA detects ADAM10sa and ADAM17sa presence in wild-type and absence in ADAM10 -/- and ADAM17 -/- MEF lysates, respectively. ADAM10 +/- , ADAM10 -/- ( A, C, E ) , ADAM17 +/+ and ADAM17 -/- ( B, D, F ) MEFs were activated with PMA/Ionomycin and lysed. The 150 μL Tris solutions of 2 μg cell lysates, 10 μM PEPDAB substrates and 0.5% Roche/Halt protease inhibitors were incubated for 4 h at 37 o C, and the developed fluorescence was recorded hourly using TECAN fluorimeter. The presented experiments are representative of 5 performed. The enzymatic activity data are shown as pM means of duplicate measurements ± SD of the processed substrates ( A, B ). The decreased processing of PEPDAB005, 010, 011, 014 and 022 with knockout MEF lysates is significant ( ADAM10 +/- vs ADAM10 -/- MEFs and ADAM17 +/+ vs ADAM17 -/- MEFs: p<0.0001 and p<0.001, p=0.0056 and p<0.001, p=0.0016 and p=0.0025, p=0.0016 and p=0.009, and p=0.006 and p=0.004, respectively). Systematic PrAMA data using Syntherror/Sigmathreshold parameters 0.5/0.0 to 0.5/2.0 and 0.5/0.0 to 0.5/1.0 are presented as ADAM10sa AU ( C ) and ADAM17sa AU ( D ) , respectively. Standard errors of PrAMA data were 2.5% to 5.8%. True-positive ADAM10sa and ADAM17sa in ADAM10 +/- and ADAM17 +/+ MEFs, respectively, are presented as % of specific enzyme activities in wild-type MEFs, containing both the true-positive and false-positive activities, relative to ADAM10 -/- (E) and ADAM17 -/- (F) ME Fs containing only the false-positive activities, respectively.

Article Snippet: Human ADAM10 and ADAM17 siRNA (pools of three target specific siRNA), control (scrambled siRNA), siRNA transfection medium, and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Modification, Incubation, Fluorescence, Activity Assay, Knock-Out

Modified PrAMA detects decreases of ADAM10sa and ADAM17sa in human cancer cells after silencing of the corresponding enzyme genes. H441 cells were transfected with human ADAM10 siRNA, ADAM17 siRNA or scrambled siRNA or were treated with transfection reagents alone for 48 h. In some experiments, control cells were also untreated. ( A , B ) Cells were stained with PE-conjugated IgG control mAb, anti-human ADAM10 ( A ) or anti-human ADAM17 mAb ( B ) and analyzed by flow cytometry. Empty histograms represent MFI of H441 cells stained with isotype control mAb. Dark gray histograms represent MFI of H441 cells treated with scrambled siRNA and stained with PE-conjugated anti-ADAM10 ( A ) or anti-ADAM17 ( B ) mAbs. Light grey histograms represent MFI of H441 cells treated with ADAM10 ( A ) or ADAM17 ( B ) siRNA and stained with PE-conjugated anti-ADAM10 or anti-ADAM17 mAbs, respectively. Data are from a representative experiment of 8 similar performed ( Suppl. Figs. A, 4B ). In the presented experiment, ADAM10 and ADAM17 siRNA induced 90% and 45% decreases of ADAM10 and ADAM17 protein expression on H441 cell surface, respectively. ( C ) After performing transfection, 2 μg of cell lysates were tested for processing PEPDABs in the presence of 0.5% Roche/Halt protease inhibitors. Data are from one of seven similar experiments performed. They are pM means of duplicate measurements ± SD of processed substrates. Processing of PEPDAB substrates was differently decreased in H441 cells transfected with ADAM10 or ADAM17 siRNA (PEPDAB005: p=0.0023 and p=0.0077; PEPDAB010: p=0.012 and p=0.017; PEPDAB011: p=0.048 and p=0.06; and PEPDAB014: p=0.07 and p=0.0.019, respectively). The substrate processing data obtained at 4 h of incubation were analyzed using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. The resulted PrAMA ADAM10sa ( D) and ADAM17sa ( E ) AU are shown. PrAMA standard errors were 1.3% to 8.9%. Decreases of ADAM10sa and ADAM17sa in ADAM10 ( F ) and ADAM17 ( G ) siRNA transfected H441 cells, respectively, are presented as % of the specific enzyme activities in the siRNA transfected cells relative to transfection reagent-treated cells. Proportion-equation analysis of PrAMA-ADAM10sa and PrAMA-ADAM17sa of rADAM10 and rADAM17 vs the scrambled siRNA-transfected H441-cell lysates, respectively, showed that 10 μg of H441-cell lysate contained 17.0 ng of ADAM10sa and 1.65 ng of ADAM17sa.

Journal: Journal of Cancer

Article Title: Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

doi: 10.7150/jca.20779

Figure Lengend Snippet: Modified PrAMA detects decreases of ADAM10sa and ADAM17sa in human cancer cells after silencing of the corresponding enzyme genes. H441 cells were transfected with human ADAM10 siRNA, ADAM17 siRNA or scrambled siRNA or were treated with transfection reagents alone for 48 h. In some experiments, control cells were also untreated. ( A , B ) Cells were stained with PE-conjugated IgG control mAb, anti-human ADAM10 ( A ) or anti-human ADAM17 mAb ( B ) and analyzed by flow cytometry. Empty histograms represent MFI of H441 cells stained with isotype control mAb. Dark gray histograms represent MFI of H441 cells treated with scrambled siRNA and stained with PE-conjugated anti-ADAM10 ( A ) or anti-ADAM17 ( B ) mAbs. Light grey histograms represent MFI of H441 cells treated with ADAM10 ( A ) or ADAM17 ( B ) siRNA and stained with PE-conjugated anti-ADAM10 or anti-ADAM17 mAbs, respectively. Data are from a representative experiment of 8 similar performed ( Suppl. Figs. A, 4B ). In the presented experiment, ADAM10 and ADAM17 siRNA induced 90% and 45% decreases of ADAM10 and ADAM17 protein expression on H441 cell surface, respectively. ( C ) After performing transfection, 2 μg of cell lysates were tested for processing PEPDABs in the presence of 0.5% Roche/Halt protease inhibitors. Data are from one of seven similar experiments performed. They are pM means of duplicate measurements ± SD of processed substrates. Processing of PEPDAB substrates was differently decreased in H441 cells transfected with ADAM10 or ADAM17 siRNA (PEPDAB005: p=0.0023 and p=0.0077; PEPDAB010: p=0.012 and p=0.017; PEPDAB011: p=0.048 and p=0.06; and PEPDAB014: p=0.07 and p=0.0.019, respectively). The substrate processing data obtained at 4 h of incubation were analyzed using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. The resulted PrAMA ADAM10sa ( D) and ADAM17sa ( E ) AU are shown. PrAMA standard errors were 1.3% to 8.9%. Decreases of ADAM10sa and ADAM17sa in ADAM10 ( F ) and ADAM17 ( G ) siRNA transfected H441 cells, respectively, are presented as % of the specific enzyme activities in the siRNA transfected cells relative to transfection reagent-treated cells. Proportion-equation analysis of PrAMA-ADAM10sa and PrAMA-ADAM17sa of rADAM10 and rADAM17 vs the scrambled siRNA-transfected H441-cell lysates, respectively, showed that 10 μg of H441-cell lysate contained 17.0 ng of ADAM10sa and 1.65 ng of ADAM17sa.

Article Snippet: Human ADAM10 and ADAM17 siRNA (pools of three target specific siRNA), control (scrambled siRNA), siRNA transfection medium, and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Modification, Transfection, Control, Staining, Flow Cytometry, Expressing, Incubation

Modified PrAMA efficiently measures ADAM10sa and ADAM17sa in multiple human tumor-tissue specimens. ( A ) NSCLC tumor lysates contain less ADAM10 than ADAM17 protein. ADAM10 and ADAM17 were quantified in lysates of 5 human NSCLC tumor tissues using ELISAs. Presented data are means pg/10 μg lysates of ADAM10 and ADAM17 proteins ± SD of 5 tumor tissues. ( B ) NSCLC tissue lysates process high amounts of PEPDAB05, 008 and 010, moderate amounts of PEPDAB014 and 022, and low amounts of PEPDAB011 and 013. Duplicates of 150 μL of Tris-based reaction buffer supplemented with NSCLC tumor-tissue lysates (10 μg/replicate), 0.5% Roche/Halt protease inhibitors and 10 μM of PEPDABs were incubated at 37 o C, and fluorescence quantified hourly for 4 h. Data are pM means of duplicate measurements ± SD of processed substrates with the 5 tissue lysates. ( C , E ) PrAMA ADAM10sa and ( D , F ) PrAMA ADAM17sa are robust but quantitatively different. The 4 h substrate processing data were analyzed using the systematically increased Syntherror/Sigmathreshold scripts from 0.5/0.0 to 0.5/2.0. Presented data are of the individual tissue lysates. PrAMA standard errors were 2.7% to 5.4%. Proportion-equation analysis of rADAM10 and rADAM17, and the tissue lysate PrAMA-ADAM10sa and PrAMA-ADAM17sa, respectively, showed that 10 μg of these tissue lysates contained 20.0 ng of ADAM10sa and 1.25 ng of ADAM17sa.

Journal: Journal of Cancer

Article Title: Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

doi: 10.7150/jca.20779

Figure Lengend Snippet: Modified PrAMA efficiently measures ADAM10sa and ADAM17sa in multiple human tumor-tissue specimens. ( A ) NSCLC tumor lysates contain less ADAM10 than ADAM17 protein. ADAM10 and ADAM17 were quantified in lysates of 5 human NSCLC tumor tissues using ELISAs. Presented data are means pg/10 μg lysates of ADAM10 and ADAM17 proteins ± SD of 5 tumor tissues. ( B ) NSCLC tissue lysates process high amounts of PEPDAB05, 008 and 010, moderate amounts of PEPDAB014 and 022, and low amounts of PEPDAB011 and 013. Duplicates of 150 μL of Tris-based reaction buffer supplemented with NSCLC tumor-tissue lysates (10 μg/replicate), 0.5% Roche/Halt protease inhibitors and 10 μM of PEPDABs were incubated at 37 o C, and fluorescence quantified hourly for 4 h. Data are pM means of duplicate measurements ± SD of processed substrates with the 5 tissue lysates. ( C , E ) PrAMA ADAM10sa and ( D , F ) PrAMA ADAM17sa are robust but quantitatively different. The 4 h substrate processing data were analyzed using the systematically increased Syntherror/Sigmathreshold scripts from 0.5/0.0 to 0.5/2.0. Presented data are of the individual tissue lysates. PrAMA standard errors were 2.7% to 5.4%. Proportion-equation analysis of rADAM10 and rADAM17, and the tissue lysate PrAMA-ADAM10sa and PrAMA-ADAM17sa, respectively, showed that 10 μg of these tissue lysates contained 20.0 ng of ADAM10sa and 1.25 ng of ADAM17sa.

Article Snippet: Human ADAM10 and ADAM17 siRNA (pools of three target specific siRNA), control (scrambled siRNA), siRNA transfection medium, and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Modification, Incubation, Fluorescence

RTDs inhibit cellular TACE and ADAM10 sheddase activities. A and B, RTDs, but not acyclic S7 or α-defensin HNP-4, suppress TACE-mediated ectodomain shedding of TGFα in COS7 cells. Data are expressed as fold-TGFα shedding relative to constitutive TGFα release. C, RTD-1, but not S7 or HNP-4, inhibits ADAM10-dependent cleavage of BTC in COS7 cells. Data are expressed as fold BTC shedding relative to constitutive BTC release. Data in panels A--C represent mean ± S.D. of a representative experiment containing 3 technical repeats. D, RTDs inhibit rADAM10 proteolysis of its fluorogenic substrate. Enzyme reactions were performed for 30 min at 37 °C with 1 nm ADAM10 and 10 μm substrate (R&D Systems, ES010). Enzyme inhibition is expressed as percent change in substrate conversion rate relative to peptide-free controls. Data represent mean ± S.D. of 2 independent experiments containing 2 technical repeats each.

Journal: The Journal of Biological Chemistry

Article Title: Macrocyclic θ-defensins suppress tumor necrosis factor-α (TNF-α) shedding by inhibition of TNF-α–converting enzyme

doi: 10.1074/jbc.RA117.000793

Figure Lengend Snippet: RTDs inhibit cellular TACE and ADAM10 sheddase activities. A and B, RTDs, but not acyclic S7 or α-defensin HNP-4, suppress TACE-mediated ectodomain shedding of TGFα in COS7 cells. Data are expressed as fold-TGFα shedding relative to constitutive TGFα release. C, RTD-1, but not S7 or HNP-4, inhibits ADAM10-dependent cleavage of BTC in COS7 cells. Data are expressed as fold BTC shedding relative to constitutive BTC release. Data in panels A--C represent mean ± S.D. of a representative experiment containing 3 technical repeats. D, RTDs inhibit rADAM10 proteolysis of its fluorogenic substrate. Enzyme reactions were performed for 30 min at 37 °C with 1 nm ADAM10 and 10 μm substrate (R&D Systems, ES010). Enzyme inhibition is expressed as percent change in substrate conversion rate relative to peptide-free controls. Data represent mean ± S.D. of 2 independent experiments containing 2 technical repeats each.

Article Snippet: TACE and ADAM10 inhibition assays Proteolytic activities of recombinant human TACE (R&D Systems, 930-ADB, 52 kDa, full ectodomain) and ADAM10 (R&D Systems, 936-AD, 52 kDa, full ectodomain) were measured using fluorogenic substrates Mca-PLAQAV-Dpa-RSSSR-NH 2 (R&D Systems, ES003) and Mca-KPLGL-Dpa-AR-NH 2 (R&D Systems, ES010), respectively.

Techniques: Enzyme Inhibition Assay