untagged human adam10  (Addgene inc)

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) Total spectral matches assigned to ADAM10 and ADAM17 obtained by mass spectrometry for OptiPrep gradient–purified Nef + and Nef − virions produced in MOLT-3 cells. ( B ) Expression of ADAM10 and ADAM17 mRNAs in MOLT-3 cells quantified by transcriptome sequencing (RNA sequencing) as fragments per kilobase of transcript per million mapped reads (FPKM) ( n = 4). ( C ) ADAM10 surface levels on parental MOLT-3 cells and ADAM10 KO clones analyzed by flow cytometry. The gray-shaded histogram represents staining of MOLT-3 cells with an isotype control. ( D ) Western blots showing that Nef − virions produced in MOLT-3–derived ADAM10 KO clones completely lack the gp41-derived CTF and contain more gp41. ( E ) ADAM10 surface levels on parental MOLT-3 cells and on an ADAM10 KO clone stably transduced with pCX4purADAM10. ( F ) Western blots showing that the gp41-derived CTF reappears in virions after ADAM10 expression is partially restored in the virus-producing cells.

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Mass Spectrometry, Purification, Produced, Expressing, Sequencing, RNA Sequencing, Clone Assay, Flow Cytometry, Staining, Control, Western Blot, Derivative Assay, Stable Transfection, Transduction, Virus

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) Expression of ADAM10 on MOLT-3 pools obtained by fluorescent-activated cell sorting (FACS) after CRISPR-Cas9–mediated editing of the ADAM10 gene. Gray-shaded histograms represent the isotype control. ( B ) Virus growth curves showing the propagation of Nef + and Nef − HIV-1 NL4-3 in the FACS-sorted MOLT-3 pools. The pools were infected with equal amounts (0.2 ng of p24/ml) of Nef + or Nef − HIV-1 NL4-3 . ( C ) Repeat experiment showing Gag expression in ADAM10 + and ADAM10 − pools of MOLT-3 cells 10 days after infection as in (B). ( D ) Relative amounts of cell-associated HIV-1 CA in ADAM10 + or ADAM10 − pools of MOLT-3 cells 8 to 10 days after infection as in (B) ( n = 3). The values are from a densitometric analysis of the Western blots shown in (C) and fig. S2, with the values obtained for ADAM10 + pools infected with Nef + virions normalized to 1. ** P < 0.01 (two-tailed unpaired t test). ( E ) ADAM10 surface levels on FACS-sorted Jurkat E6.1 pools. ( F and G ) HIV-1 replication in the FACS-sorted Jurkat pools after infection as in (B). Virus replication was monitored by p24 enzyme-linked immunosorbent assay (ELISA) (F) and by Western blotting of cell lysates with anti-CA (G). pi, postinfection. ( H ) Relative amounts of cell-associated HIV-1 CA in ADAM10 + or ADAM10 − Jurkat pools 9 to 10 days after infection as in (B) ( n = 3). The values are from a densitometric analysis of the Western blots shown in fig. S5, with the values obtained for ADAM10 + pools infected with Nef + virions normalized to 1. ** P < 0.01 (two-tailed unpaired t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Expressing, FACS, CRISPR, Control, Virus, Infection, Western Blot, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) CD4 surface levels on purified primary CD4 + T cells after stimulation with phytohemagglutinin (PHA). The gray-shaded histogram represents the isotype control. ( B ) ADAM10 surface levels on the same cells after nucleofection of Cas9 complexed with non-targeting (NTC) sgRNA or the TS1 sgRNA targeting ADAM10 . ( C ) ADAM10 surface levels on primary CD4 + T cells from the same donor sorted into ADAM10 + and ADAM10 − pools. ( D ) Virus replication in the FACS-sorted pools from the same donor after infection with equal amounts (0.5 ng of p24/ml) of Nef + or Nef − HIV-1 NL4-3 . Virus replication was monitored by p24 ELISA. ( E ) Replication of Nef + or Nef − HIV-1 NL4-3 in FACS-sorted ADAM10 + and ADAM10 − pools of primary CD4 + T cells from another donor (donor B) infected as in (D). Infections with Nef − HIV-1 NL4-3 were performed in triplicate. ( F ) Mean p24 values in the supernatants of the ADAM10 + and ADAM10 − pools from donor B on day 13 postinfection (pi) with Nef − HIV-1 NL4-3 ( n = 3). ** P < 0.01 (two-tailed unpaired t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Purification, Control, Virus, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) CCR5 surface levels on ADAM10 + and ADAM10 − pools of Jurkat E6.1 cells after stable transduction with pCXbsrCCR5. PE, phycoerythrin. ( B and C ) Replication of Nef + or Nef − versions of NL-JRFL (B) or NL-ZM109 (C) in the same pools monitored by p24 ELISA. The ADAM10 + and ADAM10 − pools were infected with equal amounts of Nef + or Nef − versions of NL-JRFL (0.1 ng of p24/ml) or NL-ZM109 (0.2 ng of p24/ml). ( D ) Western blots showing Gag expression in the same pools on day 12 after infection. ( E ) Relative amounts of cell-associated HIV-1 CA in ADAM10 + or ADAM10 − pools of MOLT-3 cells 12 to 13 days after infection with equal amounts (0.1 ng of p24/ml) of Nef + or Nef − NL-JRFL ( n = 3). The values are from a densitometric analysis of Western blots from three experiments, with the values obtained for ADAM10 + pools infected with Nef + virions normalized to 1. ** P < 0.01 (two-tailed unpaired t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) Expression of ADAM10 on 293T subpopulations sorted for the presence or absence of ADAM10 after CRISPR-Cas9–mediated editing of the ADAM10 gene. Gray-shaded histograms represent the isotype control. ( B ) Dot blots demonstrating minimal background ZsGreen expression by MOLT-3/ZsGreen reporter cells when Nef + HIV-1 NL4-3 (200 ng of p24/ml) was added together with the entry inhibitor AMD3100 to block infection. The percentage of infected (ZsGreen-expressing) reporter cells was determined by flow cytometry. ( C ) Dot blots showing ZsGreen expression in MOLT-3/ZsGreen reporter cells after infection with equal amounts of Nef + or Nef − HIV-1 NL4-3 produced in the FACS-sorted ADAM10 + or ADAM10 − 293T subpopulations. All infections were done in duplicate. Virus replication was limited to a single cycle by adding AMD3100 16 hours after infection. ( D ) Dot blots showing that a different batch of Nef − HIV-1 NL4-3 produced in the ADAM10 + or ADAM10 − 293T subpopulations yielded similar results. SSC, side scatter. ( E ) Mean percentage + SD of reporter cells infected with Nef − HIV-1 NL4-3 ( n = 3). **** P < 0.0001 (two-tailed unpaired t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Expressing, CRISPR, Control, Blocking Assay, Infection, Flow Cytometry, Produced, Virus, Two Tailed Test

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) Western blots showing SEMA7A is more abundant in Nef-deficient virions when these are produced in the absence of ADAM10. Virions were produced by ADAM10 + and ADAM10 − MOLT-3 pools that had been infected with a vesicular stomatitis virus G protein (VSV-G)–pseudotyped, Env-deficient variant of Nef − HIV-1 NL4-3 . ( B ) Relative amounts of SEMA7A associated with Nef − HIV-1 NL4-3 virions produced by ADAM10 + or ADAM10 − MOLT-3 pools as in (A), with the values obtained for virions produced in ADAM10 + cells normalized to 1 ( n = 3). ** P < 0.01 (one-sample t test). ( C ) Western blots showing that the steady-state levels of SEMA7A in ADAM10 + and ADAM10 − MOLT-3 pools are comparable. ( D ) Relative cellular steady-state levels of SEMA7A in ADAM10 + and ADAM10 − MOLT-3 pools, with the values obtained for ADAM10 + pools normalized to 1 ( n = 3). NS, not significant ( P > 0.05) (one-sample t test). ( E ) Western blots showing that SEMA7A accumulates in Nef + virions produced in the presence of endogenous ADAM10. Virions were produced by parental MOLT-3 cells that had been infected with VSV-G–pseudotyped, Env-deficient variants of Nef + or Nef − HIV-1 NL4-3 . ( F ) Relative amounts of SEMA7A associated with Nef + or Nef − HIV-1 NL4-3 virions produced by parental MOLT-3 cells as in (E), with the values obtained for Nef − virions normalized to 1 ( n = 3). * P < 0.05 (one-sample t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Western Blot, Produced, Infection, Virus, Variant Assay

Journal: Science Advances

Article Title: The ectodomain sheddase ADAM10 restricts HIV-1 propagation and is counteracted by Nef

doi: 10.1126/sciadv.adt1836

Figure Lengend Snippet: ( A ) HIV-1 NL4-3 virions produced in MOLT-3 cells contain only mature ADAM10 lacking the inhibitory prodomain. The amino acid sequence of human ADAM10 is shown, with the putative furin cleavage site upstream of the catalytic domain highlighted in red. Peptides identified by mass spectrometry in OptiPrep gradient-purified virions that match ADAM10 are highlighted in orange. ( B ) Western blots showing that HIV-1 incorporates mature ADAM10 (mADAM10), but not the pro-domain containing immature form (proADAM10). To produce virions for analysis, ADAM10 − MOLT-3 cells stably expressing C-terminally HA-tagged ADAM10 were infected with VSV-G–pseudotyped, Env-deficient variants of HIV-1 NL4-3 encoding either WT or disrupted Nef NL4-3 . ADAM10 species in the cells and in pelleted virus samples were detected by Western blotting with anti-HA. The asterisk denotes the position of an anti-HA reactive contaminant. The experiment was performed twice, each time in triplicate. ( C ) Relative amounts of mature ADAM10 detected in Nef + and Nef − virus samples examined by Western blotting as in (B), with the values obtained for Nef − virions normalized to 1 ( n = 6). *** P < 0.001 (one-sample t test).

Article Snippet: The coding sequence (CDS) for untagged human ADAM10 (GenBank, NM_001110) was amplified from pRK5M-ADAM10 (Addgene, plasmid no. 31717) ( ) and cloned into the retroviral vector pCX4pur (GenBank, AB086386) ( ).

Techniques: Produced, Sequencing, Mass Spectrometry, Purification, Western Blot, Stable Transfection, Expressing, Infection, Virus

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: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

Techniques: Recombinant, Labeling

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: Combined solutions of 0.5% Roche and 0.5% Halt inhibitors do not affect recombinant ADAM10-mediated processing of PEPDAB substrates or PrAMA-ADAM10 sheddase activity. Processing of 5 substrates with rADAM10 ( A ) and rADAM17 ( B ) in the absence and presence of 0.5% or 1% combined inhibitors is presented. Data are pM means of duplicate measurements ± SD of processed substrates. The decreases of PEPDAB005 processing with rADAM10 and rADAM17 and the increases of PEPDAB010 processing with rADAM17 in the presence of 1% Roche/Halt inhibitors are significant (p=0.0049, p=0.0046 and p=0.0038, respectively). PrAMA-ADAM10sa data of rADAM10 processing of PEPDAB substrates in the absence ( C ) and presence of 0.5% ( D ) or 1% ( E ) combined inhibitors are presented. PrAMA-ADAM17sa data of rADAM17-processing of substrates in the absence ( F ) and presence of 0.5% ( G ) or 1% ( H ) combined inhibitors are presented. The experiments in ( A ) and ( B ) were performed using similar conditions to those presented in Fig. . PrAMA was performed using a fixed 0.5 Syntherror parameter and systematically increased Sigmathreshold parameters from 0.0 to 2.0. Data are PrAMA ADAM10sa ( C , D , E ) and PrAMA ADAM17sa ( F , G , H ) arbitrary units (AU). PrAMA standard errors were <5%.The arrow-had line in ( D ) indicates that the PrAMA specificity Sigmathreshold parameters ≥ 1.0 detects only true-positive rADAM10-ADAM10sa without any false-positive ADAM17sa.

Article Snippet: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

Techniques: Recombinant, Activity Assay

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: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

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

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 in ADAM10 -/- MEFs following restoration of ADAM10 gene. ADAM10 -/- MEFs were transfected with Lipofectamine / human ADAM10- cDNA or treated with Lipofectamine alone and incubated for 24 h. In some experiments, empty plasmid-transfected ADAM10 -/- MEFs were used as an additional control. ( A ) ADAM10 -/- MEFs transfected with human ADAM10 -cDNA express human ADAM10 on cell surface. Lipofectamine treated (empty histogram) and Lipofectamine/human ADAM10 -cDNA transfected (filled histogram) cells were stained with PE-conjugated anti-human ADAM10 and examined by flow cytometry. Cells were also stained with PE-conjugated anti-mouse ADAM10 or isotype-control mAbs, and their mean fluorescence intensities (MFIs) were similar to that of the cells treated with Lipofectamine alone and stained with PE-conjugated anti-human ADAM10 mAb (data not shown). Data are from one of two similar experiments performed. ( B ) Lysates of ADAM10 -/- MEFs transfected with human ADAM10- cDNA contain increased enzyme activities as assessed with all 7 PEPDAB substrates. Lipofectamine alone treated and Lipofectamine/human ADAM10 -cDNA transfected ADAM10 -/- MEFs were lysed. The 150 μL Tris solutions containing 2 μg cell lysates, 10 μM PEPDAB substrates and 0.5% Roche/Halt inhibitors were incubated for 4 h at 37 o C, and the developing fluorescence was recorded every hour. Data are representative of three experiments performed. They are pM means of duplicate measurements ± SD of processed substrates. The increases of PEPDAB processing with the lysates of ADAM10 -cDNA transfected cells are significant (PEPDAB010: p= 0.002; PEPDAB005, 008, 011, 013, 014 and 022: p<0.001 to p<0.0001). ( C ) PrAMA-ADAM10sa AU was obtained by analyzing the enzyme activity data at 4 h of incubation using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0 . Standard errors of PrAMA data were 1.3% to 2.6%. ( D ) True-positive ADAM10sa restoration in ADAM10 -/- MEFs is presented as % of the specific enzyme activity in ADAM10- cDNA transfected MEFs, containing both the true-positive and false-positive activities, relative to Lipofectamine-alone treated ADAM10 -/- MEFs, containing only the false-positive activity.

Article Snippet: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

Techniques: Modification, Transfection, Incubation, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Activity Assay

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: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

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

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 in human-cell and -tissue lysates exposed to ADAM10 inhibitor. H441-cell ( A ) and T2495-tissue ( B ) lysates (2 μg) were supplemented with 0.5% Roche/Halt protease inhibitors, vehicle (Control) or GI254023X (1 μM), and PEPDAB substrates (10 μM), and incubated for 4 h. The developing fluorescence was measured hourly. The presented data are pM means of duplicate measurements ± SD of processed substrates, and are representative of ten experiments performed. The GI254023X-mediated inhibitions of PEPDAB005, 010 and/or 022 processing with the lysates are significant (H441: p=0.017, 0.0041 and 0.0058; T2495: p=0.05, 0.04 and 0.08, respectively). The substrate processing data were analyzed using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. The obtained data at 4 h incubation are presented as PrAMA ADAM10sa AU of H441-cell ( C ) and T2495-tissue ( D ) lysates. PrAMA standard errors were 1.2% to 9%. Decreases of ADAM10sa in H441-cell ( E ) and T2495-tissue ( F ) lysates are presented as % of the specific enzyme activities in the control lysates relative to the GI254023X treated lysates.

Article Snippet: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

Techniques: Modification, Incubation, Fluorescence

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: Control (empty) and human ADAM10-cDNA clone plasmids were obtained from OriGene (Rockville, MD).

Techniques: Modification, Incubation, Fluorescence