• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADAM17 antibody (AB39162)

ab39162 staining human normal colon. Staining is localised to membrane.
Left panel : with primary antibody at 4 ug/ml. Right panel : isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• WB

CiteAb

Western blot - Anti-ADAM17 antibody (AB39162)

Western Blotting using Anti-ADAM17 antibody, ab39162. Publication image from Herrera, B. et al., 2017, Nat Commun, 28084316. Legend direct from paper.

TNFα reduces BMPR-II expression and promotes BMPR-II cleavage in PASMCs.(a,b) BMPR2 mRNA expression, normalized to ACTB, in human dPASMCs (a) and PAECs (b) treated with IL-1β (1 ng ml−1), IL-6 (25 ng ml−1), IL-8 (25 ng ml−1) or TNFα (1 ng ml−1) for 24 h (n=3; Student's t-test). (c,d) Representative immunoblots of BMPR-II expression in human dPASMCs (c) and PAECs (d) treated with TNFα (1 ng ml−1) for 1, 4, 8 or 24 h. Reprobed forα-tubulin to ensure equal loading. The data shown are representative of three experiments. (e) Representative confocal images of immunohistochemical staining for TNFα (turquoise) andαSMA (magenta) in lung sections from control, idiopathic and heritable PAH subjects. Nuclei were counterstained with DAPI (blue). Scale bars, 100 µm. (f) Assessment of right ventricular systolic pressure (RVSP) from Bmpr2+/+, SP-C/Tnf/Bmpr2+/+, Bmpr2+/− and SP-C/Tnf/Bmpr2+/− (n=4 per group) mice. (g) Representative immunoblots of BMPR-II expression in lungs isolated from 8 week old Bmpr2+/+ and SP-C/Tnf/Bmpr2+/+ transgenic mice. Reprobed for β-actin to ensure equal loading (n=4). (h) Representative immunoblots of BMPR-II, ADAM10 and ADAM17 in human dPASMCs transfected with DharmaFECT1 alone (DH1), siADAM10, siADAM17, combined siADAM10+siADAM17 (siADAM10/17) or non-targeting siRNA control (siCP) with or without TNFα (1 ng ml−1) treatment for 24 h. Reprobed forα-tubulin to ensure equal loading. The data shown are representative of three experiments. (i) ELISA measurement of soluble BMPR-II in conditioned media from human dPASMCs transfected with wild-type and mutant 5′-myc-tagged BMPR-II constructs and treated with TNFα (1 ng ml−1) for 24 h (n=3). One-way analysis of variance with post hoc Tukey's for multiple comparisons used in f and i. *P≤0.05, ***P≤0.001. Error bars represent mean±s.e.m. Lower molecular mass BMPR-II is indicated by an arrow.

false

• WB

CiteAb

Western blot - Anti-ADAM17 antibody (AB39162)

Western Blotting using Anti-ADAM17 antibody, ab39162. Publication image from Fröhlich, C. et al., 2015, Nat Commun, 26108729. Legend direct from paper.

Genome-wide screen identifies PACS-2 as an ADAM17 regulator(a) Cellular model system used for the genome-wide siRNA screen. 21,121 individual genes were knocked down using siRNAs and the effects on ADAM17-mediated shedding of AP-HB-EGF were measured by quantifying AP cell-surface staining after PMA stimulation. (b+c) siRNA-transfected AP-HB-EGF HT1080 (b) or AP-HB-EGF HeLa (c) cells were PMA-stimulated, and the cell medium analysed for AP activity. The fold change in AP-HB-EGF release was calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value, and finally calculating the average of all individual experiments. Data in (b) were compiled from 6 individual experiments and in (c) from 3 individual experiments, each performed in triplicate. (d) AP-HB-EGF MCF-7 cells were siRNA-transfected and stimulated with PMA (left-hand panel) or ionomycin (right-hand panel). Conditioned medium was analysed for AP activity as in (b+c). Data were compiled from 4 individual experiments, each performed in triplicate. In all cases, knockdown was confirmed by western blot. On blots,

denotes a non-specific band. Graphs show mean values ± standard error of the mean (SEM). Data were

analysed by ANOVA. **p<0.01, ***p<0.001, ****p<0.0001.

false

• WB

CiteAb

Western blot - Anti-ADAM17 antibody (AB39162)

Western Blotting using Anti-ADAM17 antibody, ab39162. Publication image from Fröhlich, C. et al., 2015, Nat Commun, 26108729. Legend direct from paper.

Genome-wide screen identifies PACS-2 as an ADAM17 regulator(a) Cellular model system used for the genome-wide siRNA screen. 21,121 individual genes were knocked down using siRNAs and the effects on ADAM17-mediated shedding of AP-HB-EGF were measured by quantifying AP cell-surface staining after PMA stimulation. (b+c) siRNA-transfected AP-HB-EGF HT1080 (b) or AP-HB-EGF HeLa (c) cells were PMA-stimulated, and the cell medium analysed for AP activity. The fold change in AP-HB-EGF release was calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value, and finally calculating the average of all individual experiments. Data in (b) were compiled from 6 individual experiments and in (c) from 3 individual experiments, each performed in triplicate. (d) AP-HB-EGF MCF-7 cells were siRNA-transfected and stimulated with PMA (left-hand panel) or ionomycin (right-hand panel). Conditioned medium was analysed for AP activity as in (b+c). Data were compiled from 4 individual experiments, each performed in triplicate. In all cases, knockdown was confirmed by western blot. On blots,

denotes a non-specific band. Graphs show mean values ± standard error of the mean (SEM). Data were

analysed by ANOVA. **p<0.01, ***p<0.001, ****p<0.0001.

false