Structured Review

Seikagaku chondroitinase abc
Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the <t>chondroitinase</t> <t>ABC</t> digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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1) Product Images from "Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc"

Article Title: Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc

Journal: Matrix Biology Plus

doi: 10.1016/j.mbplus.2021.100077

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Western Blot, Molecular Weight, Labeling

2) Product Images from "Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc"

Article Title: Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc

Journal: Matrix Biology Plus

doi: 10.1016/j.mbplus.2021.100077

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Western Blot, Molecular Weight, Labeling

3) Product Images from "Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc"

Article Title: Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc

Journal: Matrix Biology Plus

doi: 10.1016/j.mbplus.2021.100077

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5 M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN- α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4 M GuHCl extracts, pN- α1(IIA) chains were identified as a single band (as expected from Fig. 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN- α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4 M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4 M GuHCl and the 0.5 M NaCl extracts (lanes 2–4). The faint bands in the 0.5 M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Western Blot, Molecular Weight, Labeling

4) Product Images from "Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation"

Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.659428

Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.
Figure Legend Snippet: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

Techniques Used: Expressing, Clone Assay, Modification, Stable Transfection, Plasmid Preparation, Immunofluorescence

5) Product Images from "Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation"

Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.659428

Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.
Figure Legend Snippet: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

Techniques Used: Expressing, Clone Assay, Modification, Stable Transfection, Plasmid Preparation, Immunofluorescence

6) Product Images from "Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation"

Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.659428

Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study ( Nadanaka et al., 2018 ). Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.
Figure Legend Snippet: Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study ( Nadanaka et al., 2018 ). Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.

Techniques Used: Inhibition, Expressing, Modification, Incubation, Flow Cytometry, Transfection, Real-time Polymerase Chain Reaction

Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.
Figure Legend Snippet: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

Techniques Used: Expressing, Clone Assay, Modification, Stable Transfection, Plasmid Preparation, Immunofluorescence

C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.
Figure Legend Snippet: C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.

Techniques Used: Colony Assay, Concentration Assay, Cell Culture, Staining, Light Microscopy, Real-time Polymerase Chain Reaction, Generated

7) Product Images from "Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies"

Article Title: Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies

Journal: Biomolecules

doi: 10.3390/biom11040508

Characterization of the purified R-10G-binding protein by glycosidase digestion and Western blotting. Purified R-10G-binding protein (0.5–4 ng) incubated with (+) or without (−) glycosidases was subjected to SDS-PAGE on a 4–15% gradient polyacrylamide gel under non-reducing conditions. The exception was PNGase F digestion, which requires preheating under reducing conditions. Western blotting using R-10G was subsequently conducted. ( A ) Purified R-10G-binding protein from 201B7 cells was digested with PNGase F (lane 1), chondroitinase ABC (lane 2), a heparinase mixture (lane 3), α1-3/4 fucosidase (lane 4), α1-2 fucosidase (lane 5), neuraminidase (lane 6), and keratanase (lane 7), and the digests were analyzed by Western blotting using R-10G. ( B ) Purified R-10G-binding protein from 201B7 (lanes 1, 3) and Tic (lanes 2, 4) cells incubated with (+) or without (−) endo-β-galactosidase (lanes 1, 2) or keratanase II (lanes 3, 4) was analyzed by Western blotting using R-10G.
Figure Legend Snippet: Characterization of the purified R-10G-binding protein by glycosidase digestion and Western blotting. Purified R-10G-binding protein (0.5–4 ng) incubated with (+) or without (−) glycosidases was subjected to SDS-PAGE on a 4–15% gradient polyacrylamide gel under non-reducing conditions. The exception was PNGase F digestion, which requires preheating under reducing conditions. Western blotting using R-10G was subsequently conducted. ( A ) Purified R-10G-binding protein from 201B7 cells was digested with PNGase F (lane 1), chondroitinase ABC (lane 2), a heparinase mixture (lane 3), α1-3/4 fucosidase (lane 4), α1-2 fucosidase (lane 5), neuraminidase (lane 6), and keratanase (lane 7), and the digests were analyzed by Western blotting using R-10G. ( B ) Purified R-10G-binding protein from 201B7 (lanes 1, 3) and Tic (lanes 2, 4) cells incubated with (+) or without (−) endo-β-galactosidase (lanes 1, 2) or keratanase II (lanes 3, 4) was analyzed by Western blotting using R-10G.

Techniques Used: Purification, Binding Assay, Western Blot, Incubation, SDS Page

8) Product Images from "Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc"

Article Title: Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc

Journal: bioRxiv

doi: 10.1101/2021.03.26.437279

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).

Techniques Used: Western Blot, Molecular Weight, Labeling

9) Product Images from "Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc"

Article Title: Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc

Journal: bioRxiv

doi: 10.1101/2021.03.26.437279

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).

Techniques Used: Western Blot, Molecular Weight, Labeling

10) Product Images from "Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc"

Article Title: Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc

Journal: bioRxiv

doi: 10.1101/2021.03.26.437279

Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).
Figure Legend Snippet: Detection of type IIA procollagen in calf annulus fibrosus. A) In western blots, type IIA collagen antibodies (Col IIA) also identified the pN-α1(IIA) chain and the slower moving, pro-α1(IIA) collagen chain in the 0.5M NaCl extract (acid soluble) of calf AF (lane 3), with faint reactivity to the pro-α1(IIA) chain in the acid insoluble fraction (lane 4). No pN-α1(IIA) chains were detected in the chondroitinase ABC digests of both AF and NP (lane 1, 5). In 4M GuHCl extracts, pN-α1(IIA) chains were identified as a single band (as expected from Figure 2 ). B) The type II collagen antibody (1C10) revealed an abundance of, pN-α1(IIB) collagen chains in the chondroitinase digest (lanes 1, and 5) as well as the 4M Gu HCl extract. Pro-α1(IIB) chains were also observed in the 4M GuHCl and the 0.5 M NaCl extracts (lanes 2-4). The faint bands in the 0.5M NaCl soluble and insoluble extracts marked with a white asterisk (*) are in the same molecular weight range as pN-α1(IIA) and pro-α1(IIA) chain bands. These extraction results indicate differences in type IIA collagen molecular assembly or differential interaction of this collagen with other matrix components. Note the line diagrams underneath the western blots shown in (A) and (B) indicating the (pro)collagen forms identified in the labeled protein bands. Red line (IIA amino propeptide containing exon 2-encoded cysteine-rich domain), blue line (IIB amino propeptide devoid of the cysteine-rich domain), black line (helical domain), black loop (carboxy propeptide).

Techniques Used: Western Blot, Molecular Weight, Labeling

11) Product Images from "Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes"

Article Title: Establishment and characterization of Drosophila cell lines mutant for heparan sulfate modifying enzymes

Journal: Glycobiology

doi: 10.1093/glycob/cwz020

Anion exchange chromatography of HS from wild-type and Hsepi cells. Metabolically 35 S-labeled GAGs isolated from wild-type (WT, open circles) and Hsepi ” and were digested with Chondroitinase ABC prior to analysis. The samples (15,000 cpm each) from medium ( A ) and cell fractions ( B ) were applied to a Mono Q column eluted with a liner gradient of 0.25–1.5 M NaCl in NaAc buffer, pH 4.5. Eluted fractions (0.5 mL) were mixed with scintillation cocktail and assayed for radioactivity. “CS” indicates degraded chondroitin sulfate disaccharides. The dashed line indicates the salt gradient. This figure is available in black and white in print and in color at Glycobiology online.
Figure Legend Snippet: Anion exchange chromatography of HS from wild-type and Hsepi cells. Metabolically 35 S-labeled GAGs isolated from wild-type (WT, open circles) and Hsepi ” and were digested with Chondroitinase ABC prior to analysis. The samples (15,000 cpm each) from medium ( A ) and cell fractions ( B ) were applied to a Mono Q column eluted with a liner gradient of 0.25–1.5 M NaCl in NaAc buffer, pH 4.5. Eluted fractions (0.5 mL) were mixed with scintillation cocktail and assayed for radioactivity. “CS” indicates degraded chondroitin sulfate disaccharides. The dashed line indicates the salt gradient. This figure is available in black and white in print and in color at Glycobiology online.

Techniques Used: Chromatography, Metabolic Labelling, Labeling, Isolation, Radioactivity

12) Product Images from "Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice"

Article Title: Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0213660

Sulfation of chondroitin sulfate proteoglycans from femoral head cartilage. Sulfation of proteoglycans was determined by HPLC disaccharide analysis after digestion by chondroitinase ABC and ACII of chondroitin sulfate proteoglycans from the femoral head cartilage of wild-type (WT) and Impad1 D175N/D175N mice at birth. In parallel the same analysis was performed also in the Impad1 knock-out ( Impad1 -/- ) mouse studied by Frederick [ 19 ]. The amount of non sulfated disaccharide (ΔDi-0S) relative to the total amount of disaccharides (ΔDi-0S, ΔDi-4S and ΔDi-6S) is significantly increased in mutant mice compared to the wild-types indicating proteoglycan undersulfation. Interestingly, the level of proteoglycan undersulfation in mutants is similar to the Frederick’s knock-out mouse. Three mice per group were used; data are reported as mean ± SD (Student’s t-test, *p
Figure Legend Snippet: Sulfation of chondroitin sulfate proteoglycans from femoral head cartilage. Sulfation of proteoglycans was determined by HPLC disaccharide analysis after digestion by chondroitinase ABC and ACII of chondroitin sulfate proteoglycans from the femoral head cartilage of wild-type (WT) and Impad1 D175N/D175N mice at birth. In parallel the same analysis was performed also in the Impad1 knock-out ( Impad1 -/- ) mouse studied by Frederick [ 19 ]. The amount of non sulfated disaccharide (ΔDi-0S) relative to the total amount of disaccharides (ΔDi-0S, ΔDi-4S and ΔDi-6S) is significantly increased in mutant mice compared to the wild-types indicating proteoglycan undersulfation. Interestingly, the level of proteoglycan undersulfation in mutants is similar to the Frederick’s knock-out mouse. Three mice per group were used; data are reported as mean ± SD (Student’s t-test, *p

Techniques Used: High Performance Liquid Chromatography, Mouse Assay, Knock-Out, Mutagenesis

13) Product Images from "GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain"

Article Title: GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain

Journal: Scientific Reports

doi: 10.1038/s41598-019-40901-2

GlcNAc6ST3 in oligodendrocytes is a major sulfotransferase of R-10G-reactive KS in the cerebral cortex of adult mice. ( a , b ) Expression of the R-10G KS epitope in the 1% Triton-soluble fractions prepared from the cerebral cortex of adult WT mice and GlcNAc6ST1, 2, 3, and 4 single KO, and GlcNAc6ST1, 3 doubly-deficient (DKO) mice. The samples were pretreated with the chondroitinase ABC before western blotting. Representative results are shown (n = 3 in a , n = 2 in b ). Densitometric quantitative analysis was used to measure the intensities of the bands in b ( open arrowhead ). The numbers denote average values relative to WT. N.D., not detected. ß-Actin was used as a loading control. ( c ) RNA-seq transcriptome results of the genes of each GlcNAc6ST family member in indicated cell types are shown. Cells were prepared from the cerebral cortex of adult mice 49 . Columns with mean values lesser than the threshold value are indicated by # 49 . Full-length blot images are presented in Supplementary Fig. S8 .
Figure Legend Snippet: GlcNAc6ST3 in oligodendrocytes is a major sulfotransferase of R-10G-reactive KS in the cerebral cortex of adult mice. ( a , b ) Expression of the R-10G KS epitope in the 1% Triton-soluble fractions prepared from the cerebral cortex of adult WT mice and GlcNAc6ST1, 2, 3, and 4 single KO, and GlcNAc6ST1, 3 doubly-deficient (DKO) mice. The samples were pretreated with the chondroitinase ABC before western blotting. Representative results are shown (n = 3 in a , n = 2 in b ). Densitometric quantitative analysis was used to measure the intensities of the bands in b ( open arrowhead ). The numbers denote average values relative to WT. N.D., not detected. ß-Actin was used as a loading control. ( c ) RNA-seq transcriptome results of the genes of each GlcNAc6ST family member in indicated cell types are shown. Cells were prepared from the cerebral cortex of adult mice 49 . Columns with mean values lesser than the threshold value are indicated by # 49 . Full-length blot images are presented in Supplementary Fig. S8 .

Techniques Used: Mouse Assay, Expressing, Western Blot, RNA Sequencing Assay

Expression and localization of R-10G-reactive keratan sulfate/chondroitin sulfate proteoglycans in the cerebral cortex of adult mice. ( a , b ) R-10G monoclonal antibody recognizes GlcNAc-6-sulfated keratan sulfate (KS) 18 , 19 . Expression of the R-10G KS epitope in the 1% Triton-soluble fractions prepared from the cerebral cortex in adult wild-type (WT) mice is shown with or without pretreatments with KS-degrading enzymes ( a ) or chondroitinase ABC. ( b ) R-10G-reactive band signals were eliminated by endo-ß-galactosidase or keratanase pretreatment. ß-Actin was used as a loading control. ( c ) Brain sections from adult WT mice were immunostained with R-10G ( red ) followed by NeuroTrace Nissl staining ( blue ). The NeuroTrace blue-fluorescent Nissl stain was used to visualize neurons. A representative confocal microscope image of the cerebral cortex is shown (n = 3). R-10G staining signals in pericellular ( arrowheads ) and intercellular spaces ( asterisk ) were detected. Full-length blot images are presented in Supplementary Fig. S8 . Scale bar: 20 µm.
Figure Legend Snippet: Expression and localization of R-10G-reactive keratan sulfate/chondroitin sulfate proteoglycans in the cerebral cortex of adult mice. ( a , b ) R-10G monoclonal antibody recognizes GlcNAc-6-sulfated keratan sulfate (KS) 18 , 19 . Expression of the R-10G KS epitope in the 1% Triton-soluble fractions prepared from the cerebral cortex in adult wild-type (WT) mice is shown with or without pretreatments with KS-degrading enzymes ( a ) or chondroitinase ABC. ( b ) R-10G-reactive band signals were eliminated by endo-ß-galactosidase or keratanase pretreatment. ß-Actin was used as a loading control. ( c ) Brain sections from adult WT mice were immunostained with R-10G ( red ) followed by NeuroTrace Nissl staining ( blue ). The NeuroTrace blue-fluorescent Nissl stain was used to visualize neurons. A representative confocal microscope image of the cerebral cortex is shown (n = 3). R-10G staining signals in pericellular ( arrowheads ) and intercellular spaces ( asterisk ) were detected. Full-length blot images are presented in Supplementary Fig. S8 . Scale bar: 20 µm.

Techniques Used: Expressing, Mouse Assay, Staining, Microscopy

14) Product Images from "The exostosin family of glycosyltransferases: mRNA expression profiles and heparan sulphate structure in human breast carcinoma cell lines"

Article Title: The exostosin family of glycosyltransferases: mRNA expression profiles and heparan sulphate structure in human breast carcinoma cell lines

Journal: Bioscience Reports

doi: 10.1042/BSR20180770

Molecular analysis of HS chains ( A ) 35 S-labeled glycosaminoglycans were purified from the surface/ECM of MCF10A, MCF7, MDA-MB-231, and HCC38 cells as described in ‘Experimental’ section. The resulting labeled polysaccharides were digested with chondroitinase ABC and subjected to chromatography on a Superose 6 column. The retarded components eluted at approximately 21–23 ml, correspond to materials that were degraded by chondroitinase ABC. The elution positions of molecular weight standards are indicated by arrows, as in [ 37 , 38 ]. ( B ) Western blot analysis of HPSE protein expression in cell lysates of the different cell lines as indicated. The image shows one representative result out of two independent experiments. ( C ) Quantitation of the active HPSE 50-kDa band. Expression was normalized to β-actin and the expression in breast cancer cell lines was expressed relative to the non-tumorigenic MCF10A expression that was set to 1. Values are mean ± S.D. of two separate experiments.
Figure Legend Snippet: Molecular analysis of HS chains ( A ) 35 S-labeled glycosaminoglycans were purified from the surface/ECM of MCF10A, MCF7, MDA-MB-231, and HCC38 cells as described in ‘Experimental’ section. The resulting labeled polysaccharides were digested with chondroitinase ABC and subjected to chromatography on a Superose 6 column. The retarded components eluted at approximately 21–23 ml, correspond to materials that were degraded by chondroitinase ABC. The elution positions of molecular weight standards are indicated by arrows, as in [ 37 , 38 ]. ( B ) Western blot analysis of HPSE protein expression in cell lysates of the different cell lines as indicated. The image shows one representative result out of two independent experiments. ( C ) Quantitation of the active HPSE 50-kDa band. Expression was normalized to β-actin and the expression in breast cancer cell lines was expressed relative to the non-tumorigenic MCF10A expression that was set to 1. Values are mean ± S.D. of two separate experiments.

Techniques Used: Labeling, Purification, Multiple Displacement Amplification, Chromatography, Molecular Weight, Western Blot, Expressing, Quantitation Assay

15) Product Images from "Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung"

Article Title: Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung

Journal: Respiratory Research

doi: 10.1186/s12931-018-0854-4

Mean ± SD proportions (percent of mono-sulphated and non-sulphated [CS] disaccharides) of the mono-sulphated (∆-di-4S and ∆-di-6S) and unsulphated (∆-di-0S) CS disaccharides measured in micro-dissected fetal lung tissue from fetuses ( a ) exposed to cortisol-infusion (131d n = 6), ( c ) exposed to antenatal betamethasone treatment (Beta, 124d, n = 6) or ( e ) bilaterally adrenalectomised (ADX, 143d, n = 6) and their relevant control fetuses. All values were measured by FACE analysis FACE gels depicting the sulphation profile of CS Δ-disaccharides in the lungs of fetuses ( b ) exposed to cortisol-infusion, ( d ) exposed to antenatal betamethasone treatment or ( f ) bilaterally adrenalectomised and their relevant control fetuses. A control sample without chondroitinase ABC digestion (labelled undigested) was electrophoresed alongside each chondroitinase ABC-digested sample in order to identify non-specific bands (arrow-heads). The density of non-specific background bands (migrating at the same position as specific bands) were substracted from the density of the bands of interest
Figure Legend Snippet: Mean ± SD proportions (percent of mono-sulphated and non-sulphated [CS] disaccharides) of the mono-sulphated (∆-di-4S and ∆-di-6S) and unsulphated (∆-di-0S) CS disaccharides measured in micro-dissected fetal lung tissue from fetuses ( a ) exposed to cortisol-infusion (131d n = 6), ( c ) exposed to antenatal betamethasone treatment (Beta, 124d, n = 6) or ( e ) bilaterally adrenalectomised (ADX, 143d, n = 6) and their relevant control fetuses. All values were measured by FACE analysis FACE gels depicting the sulphation profile of CS Δ-disaccharides in the lungs of fetuses ( b ) exposed to cortisol-infusion, ( d ) exposed to antenatal betamethasone treatment or ( f ) bilaterally adrenalectomised and their relevant control fetuses. A control sample without chondroitinase ABC digestion (labelled undigested) was electrophoresed alongside each chondroitinase ABC-digested sample in order to identify non-specific bands (arrow-heads). The density of non-specific background bands (migrating at the same position as specific bands) were substracted from the density of the bands of interest

Techniques Used:

16) Product Images from "Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung"

Article Title: Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung

Journal: Respiratory Research

doi: 10.1186/s12931-018-0854-4

Mean ± SD proportions (percent of mono-sulphated and non-sulphated [CS] disaccharides) of the mono-sulphated (∆-di-4S and ∆-di-6S) and unsulphated (∆-di-0S) CS disaccharides measured in micro-dissected fetal lung tissue from fetuses ( a ) exposed to cortisol-infusion (131d n = 6), ( c ) exposed to antenatal betamethasone treatment (Beta, 124d, n = 6) or ( e ) bilaterally adrenalectomised (ADX, 143d, n = 6) and their relevant control fetuses. All values were measured by FACE analysis FACE gels depicting the sulphation profile of CS Δ-disaccharides in the lungs of fetuses ( b ) exposed to cortisol-infusion, ( d ) exposed to antenatal betamethasone treatment or ( f ) bilaterally adrenalectomised and their relevant control fetuses. A control sample without chondroitinase ABC digestion (labelled undigested) was electrophoresed alongside each chondroitinase ABC-digested sample in order to identify non-specific bands (arrow-heads). The density of non-specific background bands (migrating at the same position as specific bands) were substracted from the density of the bands of interest
Figure Legend Snippet: Mean ± SD proportions (percent of mono-sulphated and non-sulphated [CS] disaccharides) of the mono-sulphated (∆-di-4S and ∆-di-6S) and unsulphated (∆-di-0S) CS disaccharides measured in micro-dissected fetal lung tissue from fetuses ( a ) exposed to cortisol-infusion (131d n = 6), ( c ) exposed to antenatal betamethasone treatment (Beta, 124d, n = 6) or ( e ) bilaterally adrenalectomised (ADX, 143d, n = 6) and their relevant control fetuses. All values were measured by FACE analysis FACE gels depicting the sulphation profile of CS Δ-disaccharides in the lungs of fetuses ( b ) exposed to cortisol-infusion, ( d ) exposed to antenatal betamethasone treatment or ( f ) bilaterally adrenalectomised and their relevant control fetuses. A control sample without chondroitinase ABC digestion (labelled undigested) was electrophoresed alongside each chondroitinase ABC-digested sample in order to identify non-specific bands (arrow-heads). The density of non-specific background bands (migrating at the same position as specific bands) were substracted from the density of the bands of interest

Techniques Used:

17) Product Images from "LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications"

Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002971

NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).
Figure Legend Snippet: NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).

Techniques Used: Mass Spectrometry, Radioactivity

Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .
Figure Legend Snippet: Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .

Techniques Used:

Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.
Figure Legend Snippet: Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.

Techniques Used:

Effects of sialyltransferase inhibition on the structure and composition of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , chromatograms from size-exclusion HPLC on two serially coupled G2000SWxl columns of XylNapOH-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) either untreated ( solid line ) or treated with the sialyltransferase inhibitor 3F ax -peracetyl Neu5Ac ( Neu5Ac inhib ; dashed line ). F.I. , fluorescence intensity. C , proportion of HS in XylNapOH-primed GAGs from HCC70 cells and CCD-1095Sk cells treated with or without sialyltransferase inhibitor. D , relative differences in disaccharide composition (%) after chondroitinase ABC degradation between XylNapOH-primed GAGs from HCC70 cells ( red ) and CCD-1095Sk cells ( blue ) treated or untreated with sialyltransferase inhibitor (Δdisaccharide composition = disaccharide composition Neu5Ac inhib − disaccharide composition untreated ). The bars are the means ± S.D., where n = 3.
Figure Legend Snippet: Effects of sialyltransferase inhibition on the structure and composition of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , chromatograms from size-exclusion HPLC on two serially coupled G2000SWxl columns of XylNapOH-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) either untreated ( solid line ) or treated with the sialyltransferase inhibitor 3F ax -peracetyl Neu5Ac ( Neu5Ac inhib ; dashed line ). F.I. , fluorescence intensity. C , proportion of HS in XylNapOH-primed GAGs from HCC70 cells and CCD-1095Sk cells treated with or without sialyltransferase inhibitor. D , relative differences in disaccharide composition (%) after chondroitinase ABC degradation between XylNapOH-primed GAGs from HCC70 cells ( red ) and CCD-1095Sk cells ( blue ) treated or untreated with sialyltransferase inhibitor (Δdisaccharide composition = disaccharide composition Neu5Ac inhib − disaccharide composition untreated ). The bars are the means ± S.D., where n = 3.

Techniques Used: Inhibition, High Performance Liquid Chromatography, Fluorescence

18) Product Images from "LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications"

Article Title: LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002971

NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).
Figure Legend Snippet: NREs common to XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. A–C , average HCD-MS 2 spectra of the NRE precursor ions at m/z 380.00 [1−] ( A ), 476.07 [1−] ( B ), and 490.09 [1−] ( C ) observed in chondroitinase ABC-degraded XylNap-primed GAGs from both HCC70 cells and CCD-1095Sk cells. D , incorporation of [ 3 H]methyl in XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells expressed as radioactivity in dpm per μg of GAGs. E , average HCD-MS 2 spectrum of the NRE precursor ion at m/z 556.01 [1−] observed in chondroitinase AC-I– and -II–degraded XylNap-primed GAGs from HCC70 cells. The nomenclature of the fragment ion at m/z ).

Techniques Used: Mass Spectrometry, Radioactivity

Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .
Figure Legend Snippet: Relative proportions of CS/DS and HS and disaccharide composition of XylNap-primed CS/DS from HCC70 cells and CCD-1095Sk cells. A , relative proportions of GlcUA in CS/DS (CS/DS GlcUA ), IdoUA in CS/DS distributed as alternating or single IdoUA-containing units (CS/DS IdoUA_Alt/single ), IdoUA in CS/DS distributed as blocks (CS/DS IdoUA_ChB ), and HS of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells, respectively. B and C , disaccharide composition of XylNap-primed GAGs from HCC70 cells ( B ) and CCD-1095Sk cells ( C ) after depolymerization with chondroitinase ABC (total), chondroitinase AC-I and -II ( ChAC ), chondroitinase B ( ChB ), and the remaining disaccharides degraded by chondroitinase ABC but not by chondroitinase AC-I and -II and chondroitinase B ( Ch ( ABC-AC-B .

Techniques Used:

Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.
Figure Legend Snippet: Growth of HCC70 cells in the presence of enzymatically degraded XylNap-primed CS/DS from HCC70 cells. The growth of HCC70 cells after 96 h of treatment with XylNap-primed GAGs degraded with heparinase II and III ( Hep ; black ), heparinase II and III and chondroitinase ABC ( Hep + ChABC ; gray ), heparinase II and III and chondroitinase AC-I and -II ( Hep + ChAC ; blue ), or heparinase II and III and chondroitinase B ( Hep + ChB ; white ). The concentrations of the GAGs administered to the cells were de facto lower than the indicated concentrations, as the indicated concentrations correspond to the concentrations of the GAGs before enzymatic degradation. The data points are the means ± S.D., in which n = 3. UT , untreated.

Techniques Used:

Effects of sialyltransferase inhibition on the structure and composition of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , chromatograms from size-exclusion HPLC on two serially coupled G2000SWxl columns of XylNapOH-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) either untreated ( solid line ) or treated with the sialyltransferase inhibitor 3F ax -peracetyl Neu5Ac ( Neu5Ac inhib ; dashed line ). F.I. , fluorescence intensity. C , proportion of HS in XylNapOH-primed GAGs from HCC70 cells and CCD-1095Sk cells treated with or without sialyltransferase inhibitor. D , relative differences in disaccharide composition (%) after chondroitinase ABC degradation between XylNapOH-primed GAGs from HCC70 cells ( red ) and CCD-1095Sk cells ( blue ) treated or untreated with sialyltransferase inhibitor (Δdisaccharide composition = disaccharide composition Neu5Ac inhib − disaccharide composition untreated ). The bars are the means ± S.D., where n = 3.
Figure Legend Snippet: Effects of sialyltransferase inhibition on the structure and composition of XylNap-primed GAGs from HCC70 cells and CCD-1095Sk cells. A and B , chromatograms from size-exclusion HPLC on two serially coupled G2000SWxl columns of XylNapOH-primed GAGs from HCC70 cells ( A ) and CCD-1095Sk cells ( B ) either untreated ( solid line ) or treated with the sialyltransferase inhibitor 3F ax -peracetyl Neu5Ac ( Neu5Ac inhib ; dashed line ). F.I. , fluorescence intensity. C , proportion of HS in XylNapOH-primed GAGs from HCC70 cells and CCD-1095Sk cells treated with or without sialyltransferase inhibitor. D , relative differences in disaccharide composition (%) after chondroitinase ABC degradation between XylNapOH-primed GAGs from HCC70 cells ( red ) and CCD-1095Sk cells ( blue ) treated or untreated with sialyltransferase inhibitor (Δdisaccharide composition = disaccharide composition Neu5Ac inhib − disaccharide composition untreated ). The bars are the means ± S.D., where n = 3.

Techniques Used: Inhibition, High Performance Liquid Chromatography, Fluorescence

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    Seikagaku chondroitinase abc
    Chondroitinase Abc, supplied by Seikagaku, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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