monoclonal rat anti mouse adam10 ectodomain antibody  (R&D Systems)

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Representative Western blots showing membrane levels of PrP C as revealed by surface biotinylation (I; on the left) and total PrP C levels in lysates (I; on the right) as well as ADAM10 surface expression (II) of neuronally differentiated neural stem cells (NSCs) from Nestin A10 KO and littermate control mice and after genetic reintroduction of Adam10 (A10 KO + ADAM10) or nucleofection with control vector (A10 KO + Vector) into NSCs of Nestin A10 KO mice. Flotillin served as loading control. (III) Quantification of densitometric analysis of PrP C membrane levels of experimental groups mentioned above (n = 9 independent samples for controls [set to 1]; n = 9 for Nestin A10 KO; n = 8 for Nestin A10 KO + ADAM10; n = 5 for Nestin A10 KO + Vector; significance: **p = 0.0054; ## p = 0.0014; *p = 0.0336 ; # p = 0.0212). Error bars indicate SEM. ( B ) Representative immunofluorescent PrP C (green) surface staining of neuronally differentiated NSCs derived from Nestin A10 KO (without [second row] or with genetic reintroduction of ADAM10 [third row] or vector only [fourth row]) and littermate control mice (first row), respectively. Tubulin (red) was stained after permeabilization of cells to confirm neuronal differentiation of NSCs. DAPI (blue) marks nuclei. ( C ) Representative immunostaining of PrP C (green) and ADAM10 (red) in permeabilized (upper two rows) and non-permeabilized (lower three rows) murine embryonic fibroblasts (MEFs) derived from mice with a complete knockout of ADAM10 (ADAM10 KO) or wild-type mice (control). Higher resolution of white boxes is shown in the bottom row and reveals colocalization of PrP C and ADAM10 at the plasma membrane of wild-type control MEFs. Scale bars in B and C represent 10 µm. ( D ) Western blot analysis of cell-associated PrP C levels in ADAM10 knockout (A10 KO) and wild-type (wt) MEF lysates (left part: actin served as loading control). Levels of shed PrP C were assessed in cell culture media supernatants of ADAM10 knockout and wild-type MEFs by filter column concentration (conc. media) and immunoprecipitation (IP) with a PrP C -specific antibody respectively (right part). ( E ) Levels of cell-associated (neuronal lysates) and shed PrP C (IP of media supernatants) in primary neuronal cultures of prion protein knockout ( Prnp 0/0 ), wild-type (wt; C57BL/6), prion protein overexpressing ( tg a 20 ), and Nestin A10 KO mice at embryonic day 14. IgG-HC and IgG-LC mark signals for heavy and light chain of the capturing antibody POM2. DOI: http://dx.doi.org/10.7554/eLife.04260.003

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Membrane, Expressing, Control, Plasmid Preparation, Staining, Derivative Assay, Immunostaining, Knock-Out, Clinical Proteomics, Cell Culture, Concentration Assay, Immunoprecipitation

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Western blot analysis of mature ADAM10 (mADAM10) and PrP C expression in different brain homogenates from the cortex (left) and cerebellum (right) of A10 cKO and control mice at postnatal day (P) 19. Actin served as a loading control. ( B ) Immunohistochemical detection of PrP C in forebrain of P19 mice of both genotypes. Overview (top) and magnifications showing cortex (Cx) and hippocampal CA2 and CA3 regions. Overview and details of cerebellum (Cb) are shown below. Scale bars represent 100 µm (insets for Cb: 50 µm). ( C ) Quantitative RT-PCR analysis of PrP C mRNA levels in A10 cKO mice and controls at P19 (n = 5 for each genotype). GAPDH served as a control for normalization. Error bars indicate SD. ( D ) Adult age-matched A10 cKO and wild-type littermates had a comparable body size at 35 weeks of age. ( E ) Representative immunohistochemical staining of PrP C in cortex (Cx) and hippocampal CA1 region of adult (35 weeks) A10 cO and control mice. Prion protein knockout ( Prnp 0/0 ) and overexpressing mice ( tg a 20 ) served as negative and positive controls, respectively (scale bar: 100 µm). DOI: http://dx.doi.org/10.7554/eLife.04260.004

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Quantitative RT-PCR, Staining, Knock-Out

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Scheme of a mouse brain combining a qualitative representation of the Camk2a driven ADAM10 knockout strategy and information on the sampling of specimen. The site of intracerebral inoculation of mice with RML prions is indicated by the red encircled dot. Samples of frontal brain (dotted box) were taken for biochemical analysis and determination of infectivity titers (bioassay). The rest of the brain was formalin-fixed and embedded in paraffin. Coronal sections were prepared from different layers (dashed lines) with varying distance to the site of prion inoculation (as indicated by blue arrows) and assessed by immunohistochemical analysis. ( B ) Qualitative comparison of mouse genotypes (A10 cKO, controls, tg a 20 ) with regard to PrP C or PrP Sc levels, prion-associated neuropathology (including spongiosis, astrocytosis, and microglia activation) and prion infectivity titers according to brain region and time point. Reference to corresponding figures showing original data is provided. Cb = Cerebellum; Cx = Cortex; Hc = Hippocampus; Stri = Striatum; Tha = Thalamus; n.a. = not assessed; Qualities: +++ = high/strong; ++ = medium/moderate; + = low/basal; (+) = very low/weak; o = none. DOI: http://dx.doi.org/10.7554/eLife.04260.007

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Knock-Out, Sampling, Infection, Bioassay, Immunohistochemical staining, Comparison, Activation Assay

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Representative histological analysis in the thalamic brain region of terminally prion-diseased ADAM10 cKO and control mice including H&E staining and immunohistochemical detection of PrP Sc , GFAP (for detection of astrocytes), and Iba-1 (for detection of microglia). Scale bar: 100 µm. DOI: http://dx.doi.org/10.7554/eLife.04260.011

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Control, Staining, Immunohistochemical staining

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Overviews showing vacuolization (asterisks) in forebrain samples of ADAM10 cKO ( A ) and control mice ( B ) at a terminal stage of prion disease. High abundance of clusters containing tubulovesicular structures (TVS; arrows in ( A ) and ( C )) was only found in terminal ADAM10 cKO brain (5 out of 13 square grids showed clusters of TVS whereas only 1 out of 15 square grids presented with TVS in terminal wild-type control mice). Scale bars represent 500 nm ( A , B ) or 200 nm ( C ). Inset in ( C ) shows magnification of TVS. DOI: http://dx.doi.org/10.7554/eLife.04260.012

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Assessment of total PrP (no proteinase K (PK)) and PrP Sc amounts (+PK; blot is shown with short and longer exposition) by parallel replica Western blot analysis in forebrain homogenates of age-matched A10 cKO mice and littermate controls (both at 95 days post inoculation (dpi); n = 3 for each genotype) as well as terminally diseased tg a 20 mice (at 65 dpi; n = 3). Actin was detected in the undigested homogenates (no PK) and served as loading control. Densitometric quantification of relative protein amounts from two technical replicates is shown on the right. ( B ) Morphological analysis of neuropathological lesions in forebrains (showing hippocampal and cortical brain regions) of A10 cKO, littermate controls, and tg a 20 mice at the aforementioned time points (scale bars: 200 µm in overviews and 100 µm in insets and for PrP Sc ). ( C ) Biochemical assessment of candidate toxic signaling pathways showing protein levels of total Fyn (t-Fyn), phosphorylated (Tyr416) Src (p-Src) as well as total (t-Erk1/2) and phosporylated (Thr202/Tyr204) Erk1/2 (p-Erk1/2). Actin served as a loading control and for normalization (# and § indicate use of the same actins as corresponding signaling proteins were detected on the same Western blot). Quantitative densitometric analysis of relative p-Src/t-Fyn (left) and p-Erk/t-Erk ratio (right) (n.s. = not significant). ( D ) Representative Western blot analysis (left) and quantification of three technical replicates (right) of calpain levels in aforementioned samples. Levels of ADAM10 are shown in ( C ) and ( D ) to confirm the ADAM10 status. Error bars indicate SEM; *p <0.05; **p <0.01, ***p <0.001 (p values of Student's t-test are given in the main text). DOI: http://dx.doi.org/10.7554/eLife.04260.013

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Control, Protein-Protein interactions

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Representative Western blot analysis of calpain expression in non-prion infected ADAM10 cKO and control mice. In addition, premature (pADAM10), mature ADAM10 (mADAM10) and PrP C were detected. Quantification of calpain levels by densitometric analysis of ADAM10 cKO (n = 4) and control mice (n = 5) is shown below. Actin served as loading control. ( B ) Western blot analysis of spectrin (FL = full length) and spectrin breakdown products (SBDP) in prion-infected ADAM10 cKO and control mice at 95 days post inoculation (dpi; n = 3 per genotype). FL spectrin was reduced in ADAM10 cKO mice whereas calpain-dependent SBDP (marked by asterisk at 150/145 kDa) were not increased. ( C ) Western blot analysis of p35 and p25 levels in prion-infected samples mentioned in ( B ). A reduction in p35 levels was found in two out of three ADAM10 cKO mice. A band corresponding to p25 was not detectable. # indicates that blots used for calpain detection were re-probed with an antibody against p35/p25 and thus the same actin signals were used as loading controls. DOI: http://dx.doi.org/10.7554/eLife.04260.015

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Expressing, Infection, Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Representative histological analysis of spongiotic vacuolization as revealed by H&E staining of the cerebellum and brain stem of terminally prion-diseased control and ADAM10 cKO mice. Upper row shows overview and lower two rows show higher magnifications of cerebellum and brain stem (scale bars represent 100 µm). DOI: http://dx.doi.org/10.7554/eLife.04260.018

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Staining, Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ADAM10 regulates PrP C levels at the plasma membrane and releases almost full length PrP into the extracellular space. Thereby it affects (i) neurotoxicity, (ii) PrP Sc formation, and (iii) spreading of prion pathology. (i) Lack of ADAM10 (as assessed here by use of A10 cKO mice) results in elevated PrP C membrane levels. Membrane levels of PrP C (as a receptor) likely determine PrP Sc -associated neurotoxicity (as indicated by sizes of thunderbolts and skulls) and thereby incubation times with shortest survival in tg a 20 mice and reduced incubation times in A10 cKO mice compared with wild-type littermates with longest survival (order reflected by grey triangles on the left). (ii) Shed PrP, which is most efficiently produced in tg a 20 and absent in A10 cKO mice, might block formation of PrP Sc . This is reflected by the different PrP Sc amounts found in our different experimental groups (A10 cKO > wild-type littermates > tg a 20 ). The combination of increased PrP C membrane levels and PrP Sc formation in A10 cKO mice might favor increased production of membrane pores (as indicated in the middle row on the left) and neurotoxic Ca 2+ influx with possible (‘?’) involvement of calpain. (iii) Finally, spread of prion-associated pathology within the brain also seems to be affected by the levels of ADAM10 expression since tg a 20 mice showed enhanced whereas A10 cKO mice showed reduced dissemination of neuropathological features (as indicated by size of arrowheads on the right). Key references supporting this model are given in the text. Based on this model, stimulation of ADAM10 might therefore offer a treatment option. With regard to incubation times, protective effects by reducing local membrane bound PrP C amounts and by producing a protective soluble fragment able to block PrP Sc formation seem to predominate the disadvantage of increased spread by production of anchorless prions. DOI: http://dx.doi.org/10.7554/eLife.04260.019

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Clinical Proteomics, Membrane, Incubation, Produced, Blocking Assay, Expressing