mouse mab anti human syndecan 1  (Bio-Rad)

Journal: Glycobiology

Article Title: The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9.

doi: 10.1093/glycob/cwj098

Figure Lengend Snippet: Fig. 2. SDF-1 accelerates the shedding of syndecan-4 and to a lesser extent that of syndecan-1 from HeLa cells. (A) Untreated HeLa cells culture superna- tant (10, 50, 100, or 400 μl) were harvested and proteoglycans, partially purified by application to cationic membranes, were analyzed by dot blot. Synde- can-4 was detected by enhanced chemoluminescence detection, using anti-syndecan-4 5G9 mAb. The data are expressed as the amount of syndecan shed in absorbance units (AU), measured by densitometric scanning and analyzed with an Image software. An arbitrary value of 1 is attributed to the signal obtained for 50 μl of conditioned medium. Each point represents the mean ± SE of triplicate determinations. One of three individual experiments is shown. (B–D) HeLa cells were incubated or not for 18 h with PMA (0.5 μM) or SDF-1α at 0.5, 2.5, 10, 50, and 125 nM. (E) MDMs were incubated or not for 2 h with PMA (0.5 μM) or SDF-1α (125 nM). CD44 (B), syndecan-4 (C and E), and syndecan-1 (D and E) were detected using respectively anti-CD44, anti- syndecan-4 5G9, and anti-syndecan-1 BB4 mAbs. The data are expressed as the amount of syndecan shed, in relative absorbance units to the value given by the untreated cells. Each point represents the mean ± SE of triplicate determinations. One of three individual experiments is shown (significantly differ- ent from untreated control: *p < 0.001; **p < 0.01).

Article Snippet: The membrane was then blocked by a 1-h incubation at 37°C in PBS supplemented with 0.5% BSA, 3% non-fat dry milk, and 0.5% Tween 20, washed twice with PBS/0.5% BSA supplemented with 0.5% Tween 20 (all from Sigma Aldrich) and then incubated overnight at 4°C, with anti-syndecan-1 mAb BB4 (mouse IgG1, specific for the ectodomain of syndecan-1 of human origin, Serotec, Oxford, UK), antisyndecan-4 mAb, anti-CD44 mAb, or with their respective isotypes (mouse IgG1 or IgG2a both from Pharmingen) (all at 0.1 μg/ml).

Techniques: Purification, Dot Blot, Software, Incubation, Control

Journal: The Journal of Cell Biology

Article Title: Growth factor–induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

doi: 10.1083/jcb.200508010

Figure Lengend Snippet: FGF2 induces shedding of syndecan-1, and an “unsheddable” syndecan-1 makes FGF2 responses PIPLC resistant. (A) PANC-1 cells were treated with or without 2 ng/ml FGF2 for 30 min. After washing, trypsin was used to specifically release syndecan-1 ectodomains. Half of each sample was digested with 8 mU/ml heparinase III and 0.1 U/ml chondroitinase ABC. Syndecan-1 ectodomains were detected by Western blotting with mAb B-B4. Exposure times for the first and second lanes were longer than for the third and fourth lanes. (B) PANC-1 cells were stably transfected with expression constructs for wild-type mouse syndecan-1, an engineered variant of mouse syndecan-1 that replaces the cleavage sequence required for shedding with a heterologous one, or empty expression vector. Multiple clones of each type were expanded and examined by immunocytochemistry (not depicted) and Western blotting using mouse-specific syndecan-1 mAb 281.2. Western blot results from three representative clones are shown. Lanes 1 and 2, sham transfected; lanes 3 and 4, wild-type syndecan-1; lanes 5 and 6, cleavage mutant syndecan-1. (C) PANC-1 cells stably transfected with wild-type or cleavage mutant mouse syndecan-1 (from B) were treated with 2 ng/ml FGF2 for 30 min. After rinsing, cells were treated with trypsin as in A, and the released material was digested with 8 mU/ml heparinase III and 0.1 U/ml chondroitinase ABC. Syndecan-1 core protein was measured as in B. (A–C) Arrowheads show positions of molecular mass standards (in kD). (D) The three clones shown in B were tested for MAPK activation 1 h after the addition of 1 ng/ml FGF2. For both the sham-transfected and wild-type syndecan-1–transfected clones, pretreatment with 1 U/ml PIPLC (for 1 h) dramatically reduced FGF signaling (P < 0.02 in both cases; asterisks), whereas in the clone-expressing cleavage mutant syndecan-1, no significant reduction was seen. Data are from triplicate cultures for each condition and are normalized to loading controls. Error bars represent SEM. Y axis is measured in arbitrary units.

Article Snippet: Heparinase III and chondroitinase ABC were purchased from Seikagaku; GM6001 (Galardin) was obtained from BIOMOL Research Laboratories, Inc.; Texas red–conjugated goat anti–mouse IgG antibodies were obtained from Jackson ImmunoResearch Laboratories; anti-p42/44 ERK (anti-ERK1/ERK2) was purchased from Promega or Cell Signaling Technology (similar results were obtained with either reagent); PIPLC was obtained from Invitrogen; recombinant human FGF2 was purchased from R&D Systems; bovine FGF2 was isolated from cow brain ( ); mouse anti–human syndecan-1 mAb (B-B4) was purchased from Serotec; anti–β-tubulin (D-10) was obtained from Santa Cruz Biotechnology, Inc.; and monoclonal anti-myc (9E10) was purchased from Covance, Inc.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Construct, Variant Assay, Sequencing, Plasmid Preparation, Clone Assay, Immunocytochemistry, Mutagenesis, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Growth factor–induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

doi: 10.1083/jcb.200508010

Figure Lengend Snippet: Inhibition of metalloproteinases protects cells from PIPLC inhibition of the FGF2 response. (A) Metalloproteinase inhibitors block FGF2-induced shedding of syndecan-1. PANC-1 cells were treated with or without 1μM GM6001 or 500 ng/ml TIMP-3 for 1 h and with 2 ng/ml FGF2 for 30 min. Syndecan-1 remaining on cell surfaces was released with trypsin, concentrated, digested with heparinase and chondroitinase, and quantified by Western blotting with mAb B-B4 as in . Arrows show molecular mass markers. (B) Metalloproteinase inhibition makes long-term FGF2 responses of tumor cells PIPLC insensitive, whereas the FGF2 responses of a nontumor cell line are already insensitive to PIPLC. PANC-1 cells, MDA-MB-468 breast carcinoma cells, and C2C12 mouse myoblasts were treated for 1 h with 1 μM GM6001, 500 ng/ml TIMP-3, or no protease inhibitor as indicated. Cells were then cultured for 1 h in the presence of 1 ng/ml FGF2 or no growth factor (control). FGF2 + PIPLC cells were also exposed to 1 U/ml PIPLC during both the first and second hours of incubation. Cell lysates were probed for p42/44 ERK activation as in – .

Article Snippet: Heparinase III and chondroitinase ABC were purchased from Seikagaku; GM6001 (Galardin) was obtained from BIOMOL Research Laboratories, Inc.; Texas red–conjugated goat anti–mouse IgG antibodies were obtained from Jackson ImmunoResearch Laboratories; anti-p42/44 ERK (anti-ERK1/ERK2) was purchased from Promega or Cell Signaling Technology (similar results were obtained with either reagent); PIPLC was obtained from Invitrogen; recombinant human FGF2 was purchased from R&D Systems; bovine FGF2 was isolated from cow brain ( ); mouse anti–human syndecan-1 mAb (B-B4) was purchased from Serotec; anti–β-tubulin (D-10) was obtained from Santa Cruz Biotechnology, Inc.; and monoclonal anti-myc (9E10) was purchased from Covance, Inc.

Techniques: Inhibition, Blocking Assay, Western Blot, Protease Inhibitor, Cell Culture, Incubation, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Growth factor–induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

doi: 10.1083/jcb.200508010

Figure Lengend Snippet: MMP7 is activated by FGF2, causes syndecan-1 shedding, and is sufficient to make FGF responses PIPLC sensitive. (A) Exogenous MMP7 causes shedding of syndecan-1. PANC-1 cells were treated with or without 1 μg/ml MMP7 for 30 min. The media were collected, concentrated, and digested with 8 mU/ml heparinase III and 0.1 U/ml chondroitinase ABC followed by SDS-PAGE and Western blotting with mAb B-B4. Syndecan-1 is seen as a high molecular mass smear that is converted by GAGases to a band of ∼80 kD apparent size. (B) When PANC-1 cells are pretreated with MMP7, even short-term responses to FGF2 become PIPLC sensitive. The responses of PANC-1 cells to a 15-min treatment with FGF2 were measured as in A except that where indicated, cells were treated with 1 μg/ml MMP7 30 min before the addition of PIPLC (if used) and throughout the 15-min exposure to FGF2. In MMP7-treated cells, MAPK activation was strongly decreased by PIPLC (P < 0.01). (C) FGF2 induces MMP7 activation and release from the cell surface. PANC-1 cells were exposed to 2 ng/ml FGF2 for 30 min. The medium was removed, and cell surface MMP7 was released with 0.3 mg/ml heparin (in PBS). After concentrating as in A, samples were subjected to SDS-PAGE and Western blotting using a mixture of antibodies specific for human pro-MMP7 and activated MMP7. The asterisk shows the increase in released active MMP7 in response to FGF2 treatment. (A and C) Arrows on the right show positions of molecular mass standards (in kD). (D) FGF2 releases complexes of syndecan-1 and active MMP7. PANC-1 cells were treated with 5 ng/ml FGF2 for 30 min. Medium was collected and immunoprecipitated with antisyndecan-1 antibody, and precipitates were subjected to SDS-PAGE and Western blotting for activated MMP7. Controls consisted of immunoprecipitates from cells not treated with FGF2 or precipitation omitting antisyndecan antibody. The top two bands (asterisks) in the antibody-containing samples are IgG heavy and light chains. The lowest band is active MMP7 (arrow).

Article Snippet: Heparinase III and chondroitinase ABC were purchased from Seikagaku; GM6001 (Galardin) was obtained from BIOMOL Research Laboratories, Inc.; Texas red–conjugated goat anti–mouse IgG antibodies were obtained from Jackson ImmunoResearch Laboratories; anti-p42/44 ERK (anti-ERK1/ERK2) was purchased from Promega or Cell Signaling Technology (similar results were obtained with either reagent); PIPLC was obtained from Invitrogen; recombinant human FGF2 was purchased from R&D Systems; bovine FGF2 was isolated from cow brain ( ); mouse anti–human syndecan-1 mAb (B-B4) was purchased from Serotec; anti–β-tubulin (D-10) was obtained from Santa Cruz Biotechnology, Inc.; and monoclonal anti-myc (9E10) was purchased from Covance, Inc.

Techniques: SDS Page, Western Blot, Activation Assay, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Growth factor–induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

doi: 10.1083/jcb.200508010

Figure Lengend Snippet: MMP7 is required for syndecan-1 shedding. (A) Syndecan-1 shedding activity can be removed from the cell surface by treatment with heparin. PANC-1 cells were treated with or without 0.3 mg/ml heparin for 30 min. After washing twice with PBS, cells were exposed to 5 ng/ml FGF2 for 30 min. Medium was collected and subjected to heparinase and chondroitinase digestion as in A. Samples were concentrated and subjected to SDS-PAGE and Western blotting for syndecan-1, the core protein of which appears at ∼80 kD (arrows show positions of molecular mass markers in kD). Results from two independent tests are shown. (B) Pretreatment with heparin rescues the PIPLC dependence of long-term FGF signaling. PANC-1 cells were treated with heparin as in A and were tested for the PIPLC dependence of long-term (1 h) FGF2 signaling as in A. Whereas PIPLC greatly diminished the MAPK response of control cells (P < 0.01; t test), the responses of heparin-treated cells were unchanged (P > 0.4). (C) Pretreatment with a neutralizing antibody to MMP7 renders long-term FGF signaling PIPLC insensitive. PANC-1 cells were tested for the PIPLC dependence of long-term (1 h) FGF2 signaling as in A, with antiactivated MMP7 or nonimmune antibody (both at 4 μg/ml) added 1 h before FGF2. (D) Activation of MMP7 by FGF2 does not require the action of a GM6001-sensitive metalloproteinase. PANC-1 cells were exposed to 1 μM GM600l for 1 h. Then, either 5 ng/ml FGF2 or control medium was added along with 1 μM GM6001 for 30 min. Cell surface MMP7 was harvested by extraction with 0.3 mg/ml heparin (in PBS), and samples were concentrated as in A. The presence of activated MMP7 was detected by Western blotting as in D. Band densities are averaged from triplicate determinations ± SD (error bars) from equal numbers of cells and reveal a greater than twofold increase in activated MMP7 (P < 0.05; t test). (C and D) Y axis is measured in arbitrary units.

Article Snippet: Heparinase III and chondroitinase ABC were purchased from Seikagaku; GM6001 (Galardin) was obtained from BIOMOL Research Laboratories, Inc.; Texas red–conjugated goat anti–mouse IgG antibodies were obtained from Jackson ImmunoResearch Laboratories; anti-p42/44 ERK (anti-ERK1/ERK2) was purchased from Promega or Cell Signaling Technology (similar results were obtained with either reagent); PIPLC was obtained from Invitrogen; recombinant human FGF2 was purchased from R&D Systems; bovine FGF2 was isolated from cow brain ( ); mouse anti–human syndecan-1 mAb (B-B4) was purchased from Serotec; anti–β-tubulin (D-10) was obtained from Santa Cruz Biotechnology, Inc.; and monoclonal anti-myc (9E10) was purchased from Covance, Inc.

Techniques: Activity Assay, SDS Page, Western Blot, Activation Assay