PA1-1748A

antibody from Invitrogen Antibodies

Targeting: VCAN CSPG2, PG-M

Provider product page for PA1-1748A

Western blot

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Antibody data

Antibody Data
Antigen structure
References [34]
Comments [0]
Validations
Western blot [1]
Other assay [29]

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Product number: PA1-1748A - Provider product page
Provider: Invitrogen Antibodies
Product name: Versican V0, V1 Neo Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-1748A detects versican from human samples. PA1-1748A has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~70 kDa protein representing versican from mouse cumulus oocyte complexes. PA1-1748A immunogen is a synthetic peptide corresponding to residues C G G D(436) P E A A E(441) of human Versican V0/V1 Neo. This sequence is completely conserved between mouse and rat. This immunizing peptide (PEP-235) can be used for neutralization and control experiments. This polyclonal antibody has been referred to as: JSCDPE or DPEAAE.
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 2 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

VCAN protein structure - PA1-1748A shown in red.

Supportive validation

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Supportive validation

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Experimental details: Extended Data Figure 1 Histologic characteristics of early CCM lesions and the cerebellar white matter in which they form a, P7 and P8 CCM lesions in the Krit1 model. Hindbrains from Krit1 ECKO mice were harvested at P7 and P8 and sections stained with hematoxylin-eosin to detect vascular malformations. Images are representative of 4 independent experiments. Dotted lines indicate the cerebellar white matter. Arrows indicate CCM lesions. WM, white matter; GL, granular layer. b , Versican is abundant in the white matter of the P7 hindbrain, the site of primary CCM formation in the neonatal mouse model. H-E staining of the hindbrain at low (top left) and high (bottom left) magnification. Antibody staining reveals abundant versican protein in the white matter of the hindbrain (center), the site at which CCM lesions specifically appear at this timepoint in the Krit1 mouse model, and less abundant versican in the cortex. DPEAAE staining reveals a pattern of ADAMTS-mediated versican degradation that is the inverse of intact versican, i.e. higher in the cortex and lower in the white matter (right). Results representative of three independent experiments. Scale bars: top, 500 mum; bottom, 100 mum. Boxes indicate regions shown at higher magnification below. Dotted lines indicate the cerebellar white matter. WM, white matter; GL, granular layer.

Supportive validation

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Experimental details: Figure 4 ADAMTS-5 blockade does not decrease versican processing. Representative images of versikine immunoreactivity ( red ) on EDL cross-sections from mdx mice treated with IgG control ( A ; n = 3) or ADAMTS-5 mAb ( B ; n = 3); nuclei ( blue ). Versikine protein levels in EDL and soleus muscles ( C and D ; n = 5 per muscle and treatment group) and ( E ) V0 and V1 Versican gene expression in dystrophic EDL muscles following ADAMTS-5 blockade. * P = 0.01, independent t-test. N = 5 mice for versikine immunoblotting and n = 13 mice for V0 and V1 versican gene expression. Scale bar = 100 um.

Supportive validation

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Experimental details: Figure 1 Expression of ADAMTS-15, versican and versikine in prostate cancer biopsies. ( A , B ) Immunofluorescence staining of ADAMTS-15 (green) and either versican (VCAN) ( A ) or versikine ( B ) (red) in concert with DAPI staining of nuclei (blue) in representative prostate cancer biopsies of the indicated Gleason grade. Single channel and merged images are provided at high magnification (scale bar 60 um), with the merged image also shown at low magnification (scale bar 200 um), with the relative position of the magnified area demarcated by the solid blue box. ( C , D ) Higher magnification of the merged images of ADAMTS-15/VCAN/DAPI ( C ) and ADAMTS-15/Verskine/DAPI ( D ) staining of G7B sections outlined by white boxes in panels A and B , respectively, with arrows indicating areas of co-localization in cells with an acinar epithelial morphology.

Supportive validation

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Experimental details: Figure 2 Inhibitory activity of compound 4b . ( A ) Inhibition of versicanase activity. ADAMTS-4 (5.5 nM) and -5 (0.4 nM) were incubated either with compound 4b or DMSO for 2 h at 37 degC before addition of V1-5GAG (50 nM). At each time point, reactions were stopped by addition of EDTA and ADAMTS-generated versican fragments (versikine) quantified by sandwich ELISA. The relative versicanase activity is presented and 100% activity corresponds to that in the presence of DMSO alone. ( B ) Inhibition of ADAMTS-5 aggrecanase activity. Compounds (Cpd) 4b , 5b and 6 were incubated with ADAMTS-5 (1 nM) for 2 h at 37 degC before addition of aggrecan (20 mug). Following SDS-PAGE and immunoblot, fragments cleaved at the Glu392|Ala393 bond were detected by a monoclonal neoepitope antibody recognizing the new C-terminal fragment (anti-ARGSV) and analyzed by densitometric analysis. Data are presented as mean +- SEM (n = 4). * p < 0.05 and *** p < 0.001, compared to DMSO controls; ## p < 0.01, compared to the same concentration of compound 6 (Mann-Whitney test).

Supportive validation

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Experimental details: Figure 3. Characterization of tetO -Adamts5 mice. (A) Schematic representation of the genetic strategy used to generate ADAMTS5 BEC-GOF mice in which doxycycline drives inducible expression of V5-tagged ADAMTS5 specifically in BECs. (B) Co-immunostaining of V5 and PECAM (EC marker) in P10 cerebellums reveals expression of endothelial ADAMTS5-V5 in ADAMTS5 BEC-GOF mice compared with littermate controls. Results are representative of n > 3 litters. (C) Immunostaining of versican (C, left) and DPEAAE (ADAMTS-cleaved versican; C, right) in serial cerebellar sections of P10 mice. Boxed regions are shown at higher magnification to the right. Scale bars, 150 um. (D) Quantitation of versican (left) and DPEEAE (right) IntDen along the white matter tracts in ADAMTS5 BEC-GOF mice relative to littermate controls (versican: n = 10-12 from eight litters; DPEEAE: n = 9-12 from seven litters, Student''s t test). (E and F) Versican immunostaining is reduced in ADAMTS5 BEC-GOF mice to an extent comparable to Krit1 BECKO animals at P4 (E). Scale bars, 100 um. Quantitation of versican IntDen along cerebellar white matter tracts relative to Slco1c1 -Cre; Krit1 fl/+ mice (F; n = 5 from at least four litters, ANOVA + Tukey test). (G) Immunostaining for KLF4 in the cerebellar vessels of the white matter at P10. Scale bars: 50 um. Results are representative of n > 3 litters. (H) qPCR analysis of mRNA levels of Klf2 and Klf4 in ECs isolated from control and ADAMTS5 BEC-GOF P10 cerebellums ( n = 4-6 fr

Supportive validation

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Experimental details: Figure 7. Versican cleavage products increase endothelial sprouting in vitro and are detected adjacent to wild-type ECs in mature CCM lesions in vivo. (A) Schematic of the versican protein domain structure and the lentiviral constructs used to express the indicated recombinant versican peptides. The black arrow marks the canonical ADAMTS cleavage site within the GAGbeta domain of versican by which the N-terminal ""DPEEAE"" fragment is generated. The lentiviral control construct encodes an mCherry fluorescent protein. pLenti-G1 and G3 encode the N- and C-terminal globular domains, respectively, of versican. All versican peptides are V5-tagged. LTR, long term repeat; NLS, nuclear localization signal; IRES, internal ribosomal entry site; WPRE, woodchuck posttranscriptional regulatory element; LSP, long signal peptide. (B) V5-immunoblot of conditioned medium collected from WI-38 fibroblasts 48 h post-lentiviral transduction indicates recombinant G1 and G3 are being secreted. Data are representative of two independent experiments. (C) HUVEC spheroids co-cultured with lentivirally infected WI-38 fibroblasts 10 d post-sprouting assay. Bright-field micrographs are shown on top. A Canny edge detection algorithm was applied to the image mask to identify vessel walls for visualization (bottom). Scale bars, 200 um. Results are representative of at least two independent experiments. (D and E) Quantification of the mean number of sprouts and branch points per bead ( n = 125 beads per group

Supportive validation

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Experimental details: Figure S2. Combinatorial loss of ADAMTS1 and ADAMTS4 does not alter versican levels. (A) Versican immunostaining of cerebellums from P6 littermate animals. Boxed regions are shown at higher magnification to the right. Dotted lines delineate white matter. Scale bars, 150 um. (B) Quantitation of versican IntDen along white matter tracts relative to Krit1 ECKO ; Adamts1 fl/fl ; Adamts4 +/+ mice ( n = 3 or 4 from three litters, ANOVA + Tukey test). Error bars represent +- SEM. Not significant (n.s. ), P > 0.05.

Supportive validation

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Experimental details: Figure S4. Additional immunohistochemical characterization of ADAMTS5 BEC-GOF animals. Versican and DPEAAE immunostaining in adjacent serial sections of P10 cerebellums. Controls were littermate animals carrying one or two of the following alleles: the Cre transgene, the rtTA transgene, or the tetO- Adamts5-V5 allele. White arrows indicate vascular changes within the white matter tract closely resembling nascent CCM lesions. Asterisks indicate erythrocyte autofluorescence. Scale bars, 50 um.

Supportive validation

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Experimental details: Figure 1. The VCAN pathway regulates tumor cDC1s For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.celrep.2022.111201 . (A) Schematic showing versican (VCAN)-V1 functional domains and site-specific proteolysis to generate versikine (scissors represent ADAMTS proteolytic cleavage). CS, chondroitin sulphate. (B) Stromal distribution of anti-DPEAAE IHC staining in human lung cancers. DPEAAE constitutes the C terminus of versikine (chromogen, DAB; counterstain, hematoxylin). 10x objective: scale bars, 240 mum; 40x objective: scale bars, 60 mum. (C) Triple IHC staining of human lung cancers (DPEAAE, teal; XCR1, brown; CD8, purple). (D) Distribution of VCAN expression across TCGA carcinomas ( gdc.cancer.gov ), ordered on the horizontal axis by median VCAN expression. (E) Distribution of cDC1 (BATF3-DC) score across TCGA carcinomas, ordered on the horizontal axis by median measured cDC1 score. (F) Levels of correlation between cDC1 (BATF3-DC) score and VCAN expression across TCGA carcinomas. The ranked median of VCAN expression and measured cDC1 (BATF3-DC) score is shown across the x axis (1, highest; 20, lowest). Significant (q < 0.1) correlations after multiple hypothesis correction are colored red. Error bars represent the standard error of the correlation coefficient measured using Python statsmodels. (G) Generation of Vcan +/- mice through CRISPR-Cas9-based targeting of Vcan exon 3. (H) Mass cytometry of CD45 + cells from WT (LLC implanted into W

Supportive validation

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Experimental details: Figure 7 Diminished ADAMTS and reduced versican cleavage in Galnt1 s. (A) ADAMTS1, expressed in the myocardium and developing cardiac cushion tissue (white dashed lines), is decreased at E12.5 in Galnt1 -/- relative to wild type ( Galnt1 +/+ ). (B) ADAMTS5, expressed in the myocardium and hinge region of developing valve leaflets (white dashed lines), is diminished at E12.5 and E14.5 in Galnt1 -/- relative to wild type ( Galnt1 +/+ ). (C) ADAMTS20, expressed in the myocardium and developing cardiac cushion tissues (white dashed lines), is unchanged at E12.5 in Galnt1 -/- . (D) Confocal images of intact versican (versican, green), (E) cleaved versican (DPEAAE; green), and nuclei (Nu; blue) show an increase in intact versican accompanied by a decrease in cleaved versican in E12.5 OFT cushions and E14.5 AoV of Galnt1 -/- relative to wild type. (F) Fluorescent staining intensity of intact versican at E12.5 (n = 5) and E14.5 (n = 5) and cleaved (DPEAAE) versican at E12.5 (n = 4) and E14.5 (n = 4) relative to nuclei staining, and normalized to wild type (equal to 1), at E12.5 and E14.5 shows significant increase in Galnt1 -/- OFT cushion tissue. (G) qPCR analysis of expression of major Adamts genes and versican ( Vcan ) from E12.5 OFT cushion (isolated by LCM) is unchanged in Galnt1 s relative to wild type (triplicates, n = 4). ***, P < 0.001. AoV, aortic valve. OFTC, outflow tract cushion. Scale bars: A, B, C, D, E = 10 mum.