untagged human adam10  (Addgene inc)

Journal: BMB Reports

Article Title: Recombinant ADAMTS1 promotes muscle cell differentiation and alleviates muscle atrophy by repressing NOTCH1

doi: 10.5483/BMBRep.2024-0109

Figure Lengend Snippet: Inhibitory effect of recombinant ADAMTS1 on ADAM10-mediated NOTCH1 activation. (A, B) ADAMTS1 knockdown C2C12 cells were treated with recombinant ADAMTS1 protein at a concentration of 100 ng/ml. Differentiated myotubes were visualized using Jenner’s staining (A), and the myotube length was measured (B). Scale bar: 500 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Effect of ADAM10 on muscle cell differentiation. Recombinant ADAM10 protein was added at concentrations of 0.1, 1, 10, and 100 ng/ml. Differentiated myotube length was measured. ***P < 0.001. (D) Western blotting analysis of ADAM10-treated C2C12 cells. Quantification of protein expression using ImageJ software. ***P < 0.001. (E) Immunofluorescence staining of C2C12 cells. Cells were treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Scale bar: 100 μm. (F) Western blotting analysis of C2C12 cells treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Protein expression was quantified using ImageJ software. † P < 0.05, †† P < 0.01, ††† P < 0.001 compared to control group, *P < 0.05, **P < 0.01, ***P < 0.001 compared to recombinant ADAMTS1 untreated group. (G) Quantification of Hes1 mRNA expression. † P < 0.05 compared to control group, **P < 0.01 compared to the untreated group. Data are presented as the mean ± standard deviation. Statistical significance was tested using one-way ANOVA with Tukey’s post hoc test. Con, control; si-ctrl, control siRNA; si-ATS #1, ADAMTS1 siRNA #1; si-ATS #2, ADAMTS1 siRNA #2; rA, recombinant ADAMTS1.

Article Snippet: Recombinant ADAM10 was purchased from R&D systems (936-AD).

Techniques: Recombinant, Activation Assay, Knockdown, Concentration Assay, Staining, Cell Differentiation, Western Blot, Expressing, Software, Immunofluorescence, Control, Standard Deviation

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: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: SC79 induces the shedding of the RAGE ectodomain. HAECs were incubated with 10 µM SC79 for various times (5, 10, 30, and 60 min) ( n = 4) ( A ) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min ( n = 3) ( B ). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and an anti-actin antibody. To compare the size of RAGE in cell lysate and culture supernatant, untreated cell lysate (a) and conditioned media from cells treated with 10 µM SC79 for 30 min (b) were run on the same gel and immunoblotted with the RAGE antibody ( C ). The cell lysates of HAECs treated with different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min were immunoblotted with an antibody to the C-terminal domain of human RAGE and an anti-actin antibody (n = 4) ( D ). ( * p < 0.05 vs. control)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Inhibitors of AKT and ADAM10 diminish SC79-induced RAGE ectodomain shedding. HAECs were preincubated with or without MK-2206 (1 µM), GI 254023X (2 µM), or DMSO (vehicle) for 60 min. Following this, they were further incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and an anti-actin antibody. ( n = 3, * p < 0.05 vs. control, # p < 0.05 vs. SC79 treatment alone)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Control

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: AKT1 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) HAECs express all three AKT isoforms, and AKT1-, AKT2-, and AKT3-siRNAs selectively deplete each AKT isoform. HAECs were transfected with AKT1-, AKT2-, AKT3-siRNAs, or control siRNAs, and the cell lysates were immunoblotted with antibodies to AKT1, AKT2, AKT3, or actin. ( n = 3, * p < 0.05 vs. control). ( B ) SC79 activates AKT1. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT1 (Ser473) and AKT1. ( n = 3, * p < 0.05 vs. control). ( C ) AKT1 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT1-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to AKT1 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( D ) AKT1 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT1-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, AKT1, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT1-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Transfection, Control, Incubation, Knockdown

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: AKT2 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) SC79 activates AKT2. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT2 (Ser474) and AKT2. ( n = 4, * p < 0.05 vs. control). ( B ) AKT2 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT2-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to AKT2 and actin. ( n = 4, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) AKT2 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT2-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, AKT2, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT2-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Incubation, Control, Knockdown, Transfection

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: AKT3 activation is required for SC79-induced RAGE ectodomain shedding. ( A ) SC79 activates AKT3. HAECs were incubated with 10 µM SC79 for various times (1, 5, 10, and 30 min) (upper panel) or different concentrations of SC79 (0.1, 1, 5, and 10 µM) for 30 min (lower panel). The cell lysates were immunoblotted with antibodies to p-AKT3 (Ser472) and AKT3. ( n = 3, * p < 0.05 vs. control). ( B ) AKT3 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with AKT3-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to AKT3 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) AKT3 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with AKT3-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, AKT3, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with AKT3-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Incubation, Control, Knockdown, Transfection

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: SC79 induces RAGE ectodomain shedding by promoting ADAM10 cell surface translocation. ( A ) Immunofluorescence staining to evaluate the effect of SC79 on ADAM10 localization. HAECs grown in culture dishes with a coverslip were treated with SC79 (10 µM) for 10–120 min. (a) The cells on the coverslip were fixed for 10 min with 4% paraformaldehyde without permeabilization, then immunostained with an antibody to an extracellular portion of ADAM10 and examined using confocal microscopy. DAPI was used to label the nuclei of the cells. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to ADAM10 and actin. ( n = 3, * p < 0.05 vs. control). ( B ) ADAM10 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with ADAM10-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to ADAM10 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) ADAM10 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with ADAM10-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, ADAM10, and actin. ( n = 3, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with ADAM10-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Control, Knockdown, Transfection, Incubation

Journal: Scientific Reports

Article Title: AKT activation triggers Rab14-mediated ADAM10 translocation to the cell surface in human aortic endothelial cells

doi: 10.1038/s41598-025-90624-w

Figure Lengend Snippet: Rab14 is required for SC79-induced ADAM10 cell surface translocation. ( A ) Rab14 knockdown prevents SC79-induced ADAM10 cell surface translocation. HAECs grown in culture dishes with a coverslip were transfected with Rab14-siRNA or control siRNA and then incubated for 20 min with DMSO or SC79 (10 µM). (a) Cells grown on the coverslip were immunostained with an antibody to an extracellular portion of ADAM10. Representative photos and the relative fluorescence intensities are shown (scale bar: 100 μm). (b) Cell lysates from cells that were not on the coverslip in the same culture plate were immunoblotted with antibodies to Rab14 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( B ) Rab14 knockdown inhibits SC79-induced RAGE ectodomain shedding. HAECs were transfected with Rab14-siRNA or control siRNA and then incubated for 30 min with or without SC79 (10 µM). The cell lysate and culture supernatant were immunoblotted with a monoclonal antibody to the extracellular domain of human RAGE and antibodies to Rab14 and actin. ( n = 3, * p < 0.05 vs. control cells transfected with control siRNA). ( C ) Rab14 knockdown abolishes SC79’s inhibitory effect against AGE-BSA. HAECs transfected with Rab14-siRNA or control siRNA were treated for 30 min with or without SC79 (10 µM). The cells were then treated with AGE-BSA (100 µg/ml) for 24 h. The cell lysates were immunoblotted with antibodies to ICAM-1, Rab14, and actin. ( n = 4, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA; † p < 0.05 vs. control cells transfected with Rab14-siRNA)

Article Snippet: Antibodies for human RAGE (sc-80652; a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), ADAM10 (sc-28358; a mouse monoclonal antibody for Western blotting), Rab14 (sc-271401; a mouse monoclonal antibody), ICAM-1 (sc-7891), and actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Knockdown, Transfection, Control, Incubation, Fluorescence