MA3-16888

antibody from Invitrogen Antibodies

Targeting: ACAN AGC1, CSPG1, CSPGCP, MSK16

Provider product page for MA3-16888

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ELISA

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Antibody data

Antibody Data
Antigen structure
References [32]
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Validations
Immunocytochemistry [1]
Other assay [25]

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Product number: MA3-16888 - Provider product page
Provider: Invitrogen Antibodies
Product name: Aggrecan Monoclonal Antibody (BC-3)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: Purified recombinant Aggrecan Interglobular Domain (IGD) protein (Amino acids T331-G458 with C-term His-tag, expressed in E. coli), was used to validate this antibody. The recombinant protein contains cleavage sites for aggrecanases (E373-A374 in aggrecan) and matrix metalloproteinases (N341-F342 in aggrecan). The recombinant protein was cleaved in vitro using recombinant Aggrecanase. Degradation of IGD and accumulation of cleaved fragments was visualized via MEMCode reversible protein staining, followed by Western Blot using the BC-3 antibody for the specific detection of the aggrecan neoepitope. The antibody will also recognize full length IGD, as shown by ELISA, however with significantly lower affinity. MA3-16888 has successfully been used in ELISA, immunohistochemistry (frozen and FFPE) and Western blot applications. This antibody may be used to immunostain formalin-fixed, paraffin-embedded sections, as well as alcohol-fixed frozen sections and un-fixed snap frozen sections. Samples must be deglycosylated using 0.01 Units Chondroitinase ABC, 0.01 Units Keratanase and 0.0001 Units Keratanase II per 10 µg S-GAG of non-deglycosylated aggrecan for optimal epitope recognition. MA3-16888 detects aggrecan in human, bovine, feline, canine, guinea pig, equine, porcine, rabbit, rat, and sheep samples.
Reactivity: Human, Rat, Bovine, Canine, Feline, Guinea Pig, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: BC-3
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20°C

ACAN protein structure - MA3-16888 shown in red.

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

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Experimental details: ELISA analysis of Aggrecan was performed by coating Amine-binding Maleic Anhydride Activated Plates (Product # 15110) overnight with 100 æL of 2 æg/mL Aggrecan Interglobular Domain recombinant protein (Product # RP-77527). Plates were blocked for 1 hour at room temperature with StartingBlock Blocking Buffer (Product # 37538) and incubated with 100 æL per well of an Aggrecan monoclonal antibody (Product # MA3-16888) in duplicate at 25, 12.5, 6.25, 3.125, 1.563, 0.78 æg/100 æL with serial dilutions of for 2 hours at room temperature. The plate was washed and incubated with 100 æL per well of goat anti-mouse IgG-HRP secondary antibody (Product # 31430) in all test wells at 1:30,000 for 1 hour at room temperature. Detection was performed using 1-Step Ultra TMB - ELISA Substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was then stopped with 0.16M sulfuric acid. Absorbance values were read on a spectrophotometer at 450-550 nm.

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Experimental details: Figure 7 Western blot analysis. Tenascin, complement factor D and aggrecan were analyzed from pooled healthy (H-P), pooled early (E-P) and pooled late (L-P) sample sets: 50 mug of protein extract was loaded into each lane.

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Experimental details: Fig 6 Reduced Acan contributes to Hapln1a knockdown phenotypes. (A) Longitudinal section of fin rays treated with standard control morpholino (Standard control MO) and acanb ATG-blocking morpholino (Acanb MO1) or splice blocking morpholino (Acanb MO2). Immuno-staining for Acan (red) and counterstained for nuclei with To-pro (blue). Compared to the standard MO treated fins, Acanb MO treated fins show reduced staining for Acan. White arrow identifies the basal layer of epithelium; yellow arrow identifies Acan staining in the lepidotrichia, arrow head identifies the bone; m, mesenchyme; e, epithelium. Scale bar represents 20 mum. (B) Bar graph shows that regenerate length, segment length and cell proliferation are significantly reduced upon acanb knockdown using both MO1 and MO2. The mean of percent similarity for the MO treated experimental group and the corresponding control group were estimated and compared. Statistical significance was determined using the student's t -test (P

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Experimental details: Figure 4. Effect of AG on IL-1beta-induced extracellular matrix degradation in human NP cells. NP cells were stimulated by IL-1beta with or without AG treatment. Immunofluorescence images of (A) collagen-II and (B) aggrecan expression following treatment (magnification, x400). (C) The protein expression levels of aggrecan and collagen were determined western blot analysis. (D) Quantification of collagen levels. (E) Quantification of aggrecan levels. The values are presented as the mean +- standard deviation of 3 independent experiments. *P

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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).

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Experimental details: 3 Figure Temporal expression of osteogenic-, tenogenic-, and chondrogenic-related markers in direct cocultures maintained in different ratios of osteogenic supplementation in hypoxic and normoxic setups. Gene expression of (a) osteogenic-related markers, (i) runt-related transcription factor 2 ( RUNX2 ), and (ii) collagen type I alpha 1 chain ( COL1A1 ); (b) tenogenic-related markers, (i) scleraxis ( SCX ) and (ii) mohawk ( MKX ); and (c) chondrogenic-related markers, sex-determining region Y-box 9 ( SOX9 ) (i), cartilage oligomeric matrix protein ( COMP ) (ii), aggrecan ( ACAN ) (iii) and collagen type X alpha 1 chain ( COL10A1 ) was determined by RT-PCR. Expression of target genes was normalized to the basal condition (100 BM: 0 OM) of the correspondent day under normoxia and the log-fold change calculated ( n = 3, five replicates). Immunofluorescence of (d) collagen type I (COLI), (e) tenascin C, (f) aggrecan (ACAN), (g) collagen type II (COLII), and (h) collagen type X (COLX) in co-cultures and single cultures at (i) normoxia and (ii) hypoxia for 14 days in the presence of increasing ratios of OM. Scale bars = 50 mum. Fluorescence intensity ratio quantification (iii) of proteins deposition. Data are represented as means +- SD . For (a-h), statistically significant differences are shown as * p < .05; ** p < .01; *** p < .001; **** p < .0001; b and c are statistically significant in comparison with the same condition at 7 and 14 days, respectively; (d) is statistically sign

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Experimental details: 3 Figure Increased expression of SIRT3 in H 2 O 2 -induced senescent NPC. (a, b) DCFH-DA fluorescent staining results of ROS (scale bar = 100 mum). (c, d) Immunofluorescence staining results of iNOS (scale bar = 50 mum). (e, f) Western blot results of iNOS, Ac-SOD2, and SOD2. (g, h) SA-beta-Gal staining and quantitative analysis results (scale bar = 100 mum). (i, j) Immunofluorescence staining results of p16INK4a (scale bar = 50 mum). (k) Western blot results of p16INK4a and SASP. (l-o) Immunofluorescence staining results of Col 2 and MMP13 (scale bar = 50 mum). (p) Western blot results of aggrecan, Col 2, and MMP13. (q, r) Immunofluorescence staining results of SIRT3 (scale bar = 50 mum). (s, t) Western blot results of dose and time-dependent expression of SIRT3 expression under oxidative stress. Every experiment was performed three times, and the data are shown as the mean +- SD . * p < .05, ** p < .01 versus the control group. Ac-SOD2, acetylated-SOD2; Col 2, collagen type 2; DCFH-DA, dichloro-dihydro-fluorescein diacetate; iNOS, inducible nitric oxide synthase; MMP13, matrix metalloproteinase 13; ROS, reactive oxygen species; SA-beta-Gal, senescence-associated beta-galactosidase; SASP, senescence-associated secretory phenotype; SIRT3, silent information regulator 2 homolog 3; SOD2, superoxide dismutase 2

Supportive validation

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Experimental details: 4 Figure The impact of the targeted regulation of SIRT3 on H 2 O 2 -induced senescence NPC. (a, b) DCFH-DA fluorescent staining results of ROS (scale bar = 100 mum). (c, d) Immunofluorescence staining results of iNOS (scale bar = 50 mum). (e, f) Western blot results of iNOS, Ac-SOD2, and SOD2. (g, h) SA-beta-Gal staining and quantitative analysis results (scale bar = 100 mum). (i, j) Immunofluorescence staining results of p16INK4a (scale bar = 50 mum). (k) Western blot results of p16INK4a, and SASP. (l-o) Immunofluorescence staining results of Col 2 and MMP13 (scale bar = 50 mum). (p) Western blot results of aggrecan, Col 2, and MMP13. Each experiment was conducted three times, and the data are shown as the mean +- SD . * p < .05, ** p < .01. Ac-SOD2, acetylated-SOD2; Col 2, collagen type 2; DCFH-DA, dichloro-dihydro-fluorescein diacetate; iNOS, inducible nitric oxide synthase; MMP13, matrix metalloproteinase 13; NPC, nucleus pulposus cell; ROS, reactive oxygen species; SA-beta-Gal, senescence-associated beta-galactosidase; SASP, senescence-associated secretory phenotype; SIRT3, silent information regulator 2 homolog 3; SOD2, superoxide dismutase 2

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Experimental details: 6 Figure AMPK/PGC-1a signaling pathway was involved in the regulation of SIRT3 on H 2 O 2 -induced senescent NPC. (a, b) DCFH-DA fluorescent staining results of ROS (scale bar = 100 mum). (c, d) Immunofluorescence staining results of iNOS (scale bar = 50 mum). (e, f) Western blot results of iNOS, Ac-SOD2, and SOD2. (g, h) SA-beta-Gal staining and quantitative analysis results (scale bar = 100 mum). (i, j) Immunofluorescence staining results of p16INK4a (scale bar = 50 mum). (k) Western blot results of p16INK4a and SASP. (l-o) Immunofluorescence staining results of Col 2 and MMP13 (scale bar = 50 mum). (p) Western blot results of aggrecan, Col 2, and MMP13. Each experiment was conducted three times, and the data are shown as the mean +- SD . * p < .05, ** p < .01. Ac-SOD2, acetylated-SOD2; AMPK, adenosine monophosphate-activated protein kinase; Col 2, collagen type 2; DCFH-DA, dichloro-dihydro-fluorescein diacetate; iNOS, inducible nitric oxide synthase; MMP13, matrix metalloproteinase 13; NPC, nucleus pulposus cell; PGC-1a, peroxisome proliferator-activated receptor-gamma coactivator-1alpha; ROS, reactive oxygen species; SA-beta-Gal, senescence-associated beta-galactosidase; SASP, senescence-associated secretory phenotype; SIRT3, silent information regulator 2 homolog 3; SOD2, superoxide dismutase 2

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Experimental details: FIGURE 4 XH attenuated ECM degradation in IL-1beta-treated chondrocytes. (A) The immunofluorescence study of MMP-13 in IL-1beta-treated chondrocytes. (B) The summary data of fluorescence intensity of MMP-13. (C) The proteins expression of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, collagen-II, and aggrecan were detected by western blotting. (D-I) were the quantified values of tested proteins. All experiments were performed in triplicate and data are presented as the mean +- standard deviation. * p < 0.05 and ** p < 0.01. NC, negative control; Low, XH (5 muM); High, XH (20 muM).

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Experimental details: Fig. 1 The extracellular matrix expression and cholesterol efflux rate decreases in osteoarthritis (OA) chondrocytes. a) and b) Western blot analysis of aggrecan, collagen II, and peroxisome proliferator-activated receptor (PPARgamma) expression in chondrocytes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. c) The difference in cholesterol efflux rate between the control and osteoarthritis (OA) groups. d) and e) Representative immunofluorescence staining and quantification of the positive cell numbers. Chondrocytes are stained blue and the target proteins are stained red (scale bar = 100 um). DAPI, 4',6-diamidino-2-phenylindole. All p-values calculated using independent-samples t -test.

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Experimental details: Fig. 2 Effects of UTMD sv on cartilage metabolism and lipid metabolism. a) to d) Representative Western blot and quantification of aggrecan, collagen II, and PPARgamma, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in osteoarthritis (OA) chondrocytes after treatment with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV), and US-targeted simvastatin-loaded microbubble destruction (UTMD sv ). e) Representative oil red images; scale bar = 50 mum. f) Triglyceride levels were quantified in chondrocytes by measuring absorbance at 490 nm. g) The difference in cholesterol efflux rate between groups.

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Experimental details: Fig. 3 Peroxisome proliferator-activated receptor (PPARgamma) inhibitor reduces the extracellular matrix production and the cholesterol efflux rate in chondrocytes. The chondrocytes were treated with PPARgamma inhibitor. a) to d) Western blot and quantification of the relative expression levels of aggrecan, collagen II, and PPARgamma after PPARgamma inhibitor and ultrasound-targeted simvastatin-loaded microbubble destruction (UTMD SV ) intervention in osteoarthritis (OA) chondrocytes; glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as the endogenous control. e) The effect of the PPARgamma inhibitor and UTMD SV on the cholesterol efflux rate. All p-values were calculated using one-way analysis of variance.

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Experimental details: Figure 5 Proteolytic activity of ADAMTS8. A , ADAMTS8 cleaves alpha2-macroglobulin. alpha 2 M (250 nM) was incubated with either ADAMTS8 or ADAMTS1 (250 nM) for 24 h. Samples were subjected to 4 to 12% SDS-PAGE and probed with polyclonal anti-alpha 2 M antibodies. Full-length (FL) substrates and specific cleavage fragments (CF) are indicated. All reactions were performed with the WT or EQ form of each enzyme. B , activity of ADAMTS1 (500 nM), ADAMTS4 (5 nM), ADAMTS5 (5 nM), and ADAMTS8 (5-500 nM) against bovine aggrecan (667 nM). Cleavage fragments generated after either 2 or 24 h digestion (as indicated) were detected using the anti-ARGSV neoepitope antibody BC3. C , activity of ADAMTS5 (0.005-0.5 nM) and ADAMTS8 (0.5-100 nM) against aggrecan (667 nM) after 24 h digestion. Activity of 100 nM ADAMTS8 EQ is shown for comparison. Bands were detected by the antibody 2B6, recognizing the CS stubs remaining following treatment of aggrecan with chondroitinase ABC. D , activity of ADAMTS1 (1-500 nM), ADAMTS4 (5-50 nM), ADAMTS5 (5-50 nM), and ADAMTS8 (5-500 nM) against biglycan (2 muM) was investigated after 2 or 24 h digestion (as indicated) using polyclonal antibodies against biglycan. Full-length (FL) substrates and specific cleavage fragments (CF) are indicated. E and F , activity of ADAMTS1 (500 nM), ADAMTS4 (5 nM), ADAMTS5 (5 nM), and ADAMTS8 (5-500 nM) against versican V1 ( E ) and V1-5GAG ( F ) (each at 100 nM) after 2 and 24 h digestions. Bands were detected using anti-VC an

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Experimental details: Figure 3 circPhc3 is involved in the secretion of extracellular matrix and cartilage-degrading enzymes. (A) circPhc3 expression levels in chondrocytes transfected with shRNA-NC or shRNA-circPhc3 were analysed by RT-qPCR. The results showed that circPhc3 silencing was effective. ** P

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Experimental details: Figure 4 circPhc3 is involved in mechanical loading-regulated production of extracellular matrix and cartilage-degrading enzymes. Protein expression levels were (A) determined by western blotting and semi-quantified for (B) MMP-3, (C) Aggrecan and (D) Col2alpha1 in chondrocytes transfected with Mock or circPhc3 overexpression vector, followed by treatment with 10% tensile for 48 h. ** P

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Experimental details: Figure 5 miR-93-3p targeted by circPhc3 is involved in the secretion of extracellular matrix and cartilage-degrading enzymes. (A) Bioinformatics analysis demonstrated the circPhc3 had multiple binding sites for miR-93-3p. (B) Dual luciferase reporter assay results showed that the firefly luciferase activity was significantly decreased when the cells were transfected with pMIR-circPhc3 and miR-93-3p mimics compared with those transfected with pMIR-circPh3 and miR-NC. ** P

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Experimental details: Fig. 10 Chondrogenic differentiation of hMSCs cultured for 21 days on scaffolds assessed by A immunostaining of aggrecan and collagen II expression, and B western blot analysis of aggrecan, collagen II, ERK and pERK expression (n = 2). The relative expression values are given as the fold change compared to PUS/GM group. *p < 0.05, **p < 0.01 and ***p < 0.001

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Experimental details: Figure 3 miR-204-5p mRNA expression levels quantified by RT-PCR during ( A ) embryogenesis (3, 7 and 14 days post fecundation (dpf)) and ( B ) aging (2 and 6 months, 1 and 2 years old) in a zebrafish larvae in vivo model. ( C ) ERK, pERK and aggrecan protein levels assessed by western blot (top) and quantified as a relative optical density ratio vs. B-actin (down) on protein lysates from a zebrafish larvae in vivo model. (* p < 0.05; ** p < 0.005 vs. expression levels of larvae after 3 (dpf) ( A , B ) and vs. 1 year old ( C )).

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Experimental details: Fig. 4 Comparison of MSCs derived from 2D-hESCs and 3D-hESCs. a The morphology of 2D- and 3D-hESC-MSCs at different stages of differentiation. b Flow cytometry analysis revealed specific MSC surface markers (CD44, CD29, and CD105) with negative controls (CD34, CD19, and CD45) in 2D- and 3D-hESC-MSCs. c Immunostaining of differentiated 3D-hESC-MSCs expressing an adipocyte marker (FABP-4), osteocytes maker (osteocalcin), and chondrocytes marker (aggrecan)

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Experimental details: Figure 2 Changes in the ECM composition of NHDF after a three-day RPM-exposure: Immunofluorescence images of 1 g -control cells, RPM-AD and RPM-MCS of laminin ( A - C ) and fibronectin ( D - F ). Transcriptional and translational laminin analysis: Quantitative gene expression levels of LAMA3 ( G ), intracellular laminin levels ( H ) and flow cytometric analysis of laminin-labeled cells ( I ) displaying the percentage of laminin-positive cells as well as alteration of the median fluorescence intensity (MFI). Transcriptional and translational fibronectin analysis: Quantitative gene expression levels of FN1 ( J ), intracellular fibronectin levels ( K ) and flow cytometric analysis of fibronectin-labeled cells ( L ). Transcriptional and translational aggrecan analysis: Quantitative gene expression level of ACAN ( M ), intracellular aggrecan levels ( N ) and flow cytometric analysis of chondroitin sulfate-labeled cells ( O ). Transcriptional and translational osteopontin analysis: Quantitative gene expression levels of SPP1 ( P ), intracellular osteopontin levels ( Q ) and flow cytometric analysis of osteopontin-labeled cells ( R ). Full-length blots of cropped Western blot images are presented in Supplementary Fig. S1 . *p < 0.05 1 g vs. RPM; # p < 0.05 AD vs. MCS. Scale bars: 50 um.

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Experimental details: Fig. 1 Derivation of IMRCs from hESCs. a Different phase of the IMRCs derivation protocol. b Representative morphology of cells at different stages as observed by phase contrast microscopy. hEBs human embryoid bodies. Scale bar, 100 um. c A representative chromosome spread of normal diploid IMRCs with 22 pairs of autosomes and two X chromosomes. d Copy number variation (CNV) analysis by whole-genome sequencing for hESCs, primary UCMSCs and IMRCs. UCMSCs, umbilical cord mesenchymal stem cells. e Heatmap showing MSC-specific marker and pluripotency marker gene expression changes, from hESCs and hEBs to IMRCs at passages 1-5 (P1-5), and primary UCMSCs. f IMRCs' expression of MSC-specific surface markers was determined by flow cytometry. Isotype control antibodies were used as controls for gating. Like MSCs, the IMRCs are CD34 - /CD45 - /HLA-DR - /CD90 + /CD29 + /CD73 + /CD105 + cells. g Representative immunofluorescence staining of IMRCs after they were induced to undergo adipogenic differentiation (FABP-4), osteogenic differentiation (Osteocalcin), and chondrogenic differentiation (Aggrecan). Scale bar, 100 um. h Proliferation curve of IMRCs and UCMSCs at the 15th passage ( n = 5). i Distribution of cell diameters of IMRCs and UCMSCs. j Viability of IMRCs and UCMSCs in clinical injection buffer over time at 4 degC. * P < 0.05, ** P < 0.01, *** P < 0.001; data are represented as the mean +- SEM.