goat polyclonal anti human versican  (R&D Systems)

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 1 mRNA expression pattern of versican in human endometrium. (A) RT-PCR analysis shows that all versican isoforms (V0–V3) are detected in human endometrial tissue throughout the menstrual cycle. (B) Expression of versican (a) V0, (b) V1, (c) V2 and (d) V3 across different phases of the menstrual cycle was analyzed by quantitative RT-PCR (proliferative, n = 4; early secretory, n = 4; mid-secretory, n = 4; late secretory, n = 4). One-way ANOVA was used for statistical analysis.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 2 Versican protein expression in human endometrium. (A) Endometrial tissues of various menstrual cycle phases were immunostained with anti-versican polyclonal antibody specific for the V0 and V1 isoforms. (a) Early proliferative phase, (b) mid- proliferative phase, (c) late proliferative phase, (d) early secretory phase, (e) mid-secretory phase, (f) late secretory phase; (g)–(l) are higher magnification views of the area indicated in (a)–(f), respectively. (m) Glandular epithelium in the functional layer of mid-secretory endometrium. (n) Negative control for (m) in which the anti-versican antibody was replaced by isotype-matched control antibody. Versican expression was detected in both endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) throughout the menstrual cycle. The intensity of EEC versican expression was higher in the secretory phases than in the proliferative phases. Note that intense versican expression was also detected in intra-glandular products secreted from EECs and are indicated by arrowheads in (m). Scale bars = 100 µm in (a)–(f); 50 μm in (g)–(n). (B) Signal intensities of versican V1 were assessed in the immunohistochemical images of whole endometria of various menstrual cycle phases (representative images shown in A). Immunointensity scores (0, negative; 1, very weak; 2, weak; 3, moderate; 4, strong; 5, very strong) were separately given to (a) EECs and (b) ESCs. Note that immunointensity scores of (a) EECs were significantly higher in the secretory phases than in the proliferative phases, whereas those of (b) ESCs were constant throughout the menstrual cycle. In all panels, error bars represent the standard deviation. *P < 0.05; **P < 0.01; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Functional Assay, Negative Control, Control, Immunohistochemical staining, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 3 Cyclic changes in versican expression in human endometrium. (A) Lysates of isolated EECs were subjected to western blotting using the same anti-versican antibody as in Fig. 2A, which is specific for versican V0 and V1. The lower panel shows GAPDH as control. Versican V1 was detected as multiple protein signals ranging from 440 to 500 kDa. (B) Protein signal intensities for versican V1 were significantly higher in secretory phases than in proliferative phases. Error bars represent standard deviation. *P < 0.05.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Isolation, Western Blot, Control, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 4 Effects of ovarian steroid hormones on versican expression in isolated EECs. (A) EECs were isolated from the endometrial tissue of cycle day 12 and cultured for 24 h (day 1) or 72 h (day 3). Note the intense versican deposition in the extracellular space on day 3, indicating that versican secretion from EECs increased in a time-dependent manner. (a, c) and (b, d) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm. (B) Isolated EECs were treated with ethanol (EtOH), 10 nM estradiol (E2) or 10 nM E2 plus 1 µM progesterone (P4) and then immunostained. The final concentration of EtOH was 0.1% (v/v). Versican expression levels in EECs were measured by mean fluorescent intensity. The sum of the integrated densities from five areas (central, upper, lower, right and left) in the immunostained specimen is defined as the ‘versican expression level’. The versican expression level was significantly higher after treatment with E2 and P4 (E2 + P4) than after treatment with EtOH or E2 alone (n = 5). Error bars represent standard deviation. *P < 0.05.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Isolation, Cell Culture, Immunofluorescence, Concentration Assay, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 6 Differential effects of versican V1 and V3 on BeWo spheroid attachment. (A) Phase-contrast images of BeWo spheroids after 1-h incubation on a monolayer of (a) GFP-overexpressing Ishikawa cells (ISKW-GFP), (b) versican V1-overexpressing Ishikawa cells (ISKW-V1) and (c) versican V3-overexpressing Ishikawa cells (ISKW-V3). Note that all spheroids were attached to the (b) ISKW-V1 monolayer, whereas some spheroids were floating (arrowheads) over the (a) ISKW-GFP and (c) ISKW-V3 monolayers. Scale bars = 200 µm. (B) Attachment ratios to the ISKW-V1 monolayer (n = 4) were significantly higher compared with that of the control ISKW-GFP monolayer (n = 4). (C) Attachment ratios to the ISKW-V3 monolayer (n = 6) were not significantly different than those of the control (n = 6). All values are relative to the control, ISKW-GFP. In all panels, error bars represent standard deviation. *P < 0.05; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Incubation, Control, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 5 Characterization of versican V1- and V3-overexpressing Ishikawa cells. (A) Cell lysates extracted from original Ishikawa cells (Ishikawa; lane 1), platform Ishikawa cells (ISKW; lane 2), GFP- overexpressing Ishikawa cells (ISKW-GFP; lane 3) and versican V1-overexpressing Ishikawa cells (ISKW-V1; lane 4) were subjected to western blotting. ISKW possesses a specific sequence into which foreign DNA can be inserted by a recombinase. An intense protein signal at 440 kDa corresponding to versican V1 is only detectable in ISKW-V1. (B) Cell lysates from ISKW-V1 (lane 1), conditioned medium derived from ISKW-V1 (V1-CM; lane 2) and conditioned medium derived from ISKW-GFP (GFP-CM; lane 3) were subjected to western blotting using the same anti-versican antibody as in (A). Note that the protein signal at 440kDa is detected in both ISKW-V1 cell lysates (lane 1) and V1-CM (lane 2). (C) Cell lysates extracted from original Ishikawa cells (Ishikawa; lane 1), platform Ishikawa cells (ISKW; lane 2), GFP-overexpressing Ishikawa cells (ISKW-GFP; lane 3), and versican V3-overexpressing Ishikawa cells (ISKW-V3; lane 4) were subjected to western blotting using an anti-versican monoclonal antibody raised against the G3 domain common in all versican isoforms. An intense protein signal at 76 kDa corresponding to versican V3 was only detected in ISKW-V3. (D) Cell lysates from ISKW-V3 (lane1), conditioned medium derived from ISKW-V3 (V3-CM; lane2) and CM derived from ISKW-GFP (GFP-CM; lane 3) were subjected to western blotting using the same anti-versican antibody as in (C). Note that the protein signal at 76 kDa is only detectable in the ISKW-V3 cell lysates (lane 1) but not in V3-CM (lane 2). (E) ISKW-V1 cells cultured for 48 h were subjected to immunocytochemistry using the same anti-versican antibody as in (A). Note that intense immunoreactive versican is detected not only on the cell surface but also in the extracellular space (circumscribed with the dotted line). Images (a) and (b) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm. (F) ISKW-V3 cells cultured for 48 h were subjected to immunocytochemistry using anti-versican polyclonal antibody raised against the G1 domain common in all versican isoforms. Note that intense immunoreactive versican is detected exclusively on the cell surface and not in the extracellular space (circumscribed with the dotted line). Images (a) and (b) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Western Blot, Sequencing, Derivative Assay, Cell Culture, Immunocytochemistry, Immunofluorescence

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 7 Versican V1 promotion of BeWo spheroid attachment requires the presence of chondroitin sulfate side chains. (A) BeWo spheroids are incubated on the monolayer of platform Ishikawa cells (ISKW) in the presence of conditioned medium derived from GFP-overexpressing Ishikawa cells (GFP-CM) or that derived from versican V1-overexpressing Ishikawa cells (V1-CM). The attachment ratio is significantly higher in the presence of V1-CM (n = 6) than in the presence of GFP-CM (n = 6). All values are relative to the control, GFP-CM. (B) BeWo spheroids were incubated on ISKW monolayers in the presence of GFP-CM or V1-CM with or without chondroitinase ABC (ChABC) pretreatment. The attachment ratio significantly increased in the presence of V1-CM (n = 7) compared with that of GFP-CM (n = 7). Note that the attachment- promoting effect of V1-CM was completely abrogated by ChABC pretreatment (n = 7). All values are relative to the control, GFP-CM, without ChABC treatment. (C) BeWo spheroids were incubated on ISKW monolayers in the presence of V1-CM with or without hyaluronidase (Hase) pretreatment. Note that Hase pretreatment did not affect the attachment ratio that was increased by untreated V1-CM (n = 4). In all panels, error bars represent standard deviation. *P < 0.05; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Incubation, Derivative Assay, Control, Standard Deviation

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Western blots confirm increased levels of versican in the lungs of patients with pulmonary arterial hypertension (PAH). Proteoglycan extracts from lung tissue homogenates treated with the chondroitinase ABC were used. For each sample, 1.5 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, and immunoblotting for versican was performed using two different antibodies, mAb 2B1 for the C-terminal G3 domain (A) and pAb 3054 for the βGAG domain (B). Samples from 4 patients with idiopathic PAH (IPAH) were compared with age-matched unused donor lungs (control). Increased versican fragments (lanes 5, 6, and 8) were observed in subjects with IPAH compared with age-matched control subjects. C, Schematic drawing of versican splice variants and recognition sites for mAb 2B1 and pAb 3054. Versican has two globular domains, the G1 domain at the amino terminus and the G3 domain at the carboxy terminus. The splice variants vary in the GAG-attachment domains (αGAG and βGAG). C: complement regulatory region; E: epidermal growth factor–like domain; HABR: hyaluronan-binding region; Ig: immunoglobulin-like domain; L: lectin-binding domain.

Article Snippet: Two different primary antibodies were used to detect versican, a goat polyclonal antibody against βGAG domain (1∶3000, AF 3054, R&D systems) and a mouse monoclonal antibody against C-terminal G3 domain (2B1, 1∶3000, Seikagaku).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Control, Binding Assay

Journal: Oncogene

Article Title: Arylsulfatase B Regulates Versican Expression by Galectin-3 and AP-1 Mediated Transcriptional Effects

doi: 10.1038/onc.2013.483

Figure Lengend Snippet: Panels A,B,C,D. Prostate stromal cells were grown in transwells, and cells were stained following treatment with control siRNA or ARSB siRNA. Versican was immunostained with mouse monoclonal antibody and goat anti-mouse Alexa Fluor 594 (Invitrogen), and visualized. Versican immunostaining is faint in the control prostate cells (A), but intense red staining is present following ARSB silencing (B). Merged images with staining for β-actin (green) and DNA (blue) demonstrate prominent versican immunostaining. IgG control showed no red staining (not shown). Panel E. Consistent with the images above, versican protein increased from baseline of 121.0 ± 5.0 ng/mg protein to 287.5 ± 20.7 ng/mg protein in the prostate stromal cells when ARSB was silenced (p<0.001). Panel F. Similarly, in the prostate epithelial cells, versican increased from baseline of 141.6 ± 12.1 to 319.1 ± 11.9 ng/mg protein when ARSB was silenced (p<0.001). Panel G. In the ARSB deficient mice, the prostate versican protein was 217.1 ± 32.2 ng/mg protein, significantly more than in the control mouse prostate (125.9 ± 7.3 ng/mg protein; p<0.01, unpaired t-test, two-tailed). Panel H. When ARSB was silenced in the prostate stromal cells, versican mRNA expression increased more than two-fold (p<0.001). This increase was completely inhibited when galectin-3 was also silenced. Panels I, J . The combination of galectin-3 silencing and ARSB silencing nullified the increase in ARSB protein that followed ARSB silencing in the prostate stromal and epithelial cells (p<0.001). These findings demonstrate that the increased expression of versican following ARSB silencing requires galectin-3. Panel K . When prostate stromal cells were exposed to inhibitors of AP-1, identified as I1 and I2, the ARSB-knockdown induced increase in versican was reversed (p<0.001). I1 is a c-Jun mimetic peptide that impairs binding of JNK to c-Jun. I2 is an oligonucleotide-binding inhibitor that competes with c-Fos for the AP-1 oligonucleotide binding site. he inhibitors I1 and I2 inhibited the ARSB-knockdown induced increase in versican protein in the prostate stromal cells (p<0.001).

Article Snippet: Preparations were washed with PBS, blocked in 5% normal horse serum (KPL, Inc., Gaithersburg, MD) incubated overnight with goat polyclonal versican antibody (AF3054, R&D) or rabbit polyclonal galectin-3 antibody (sc-20157, SCBT) at 4°C, then washed and stained with Alex Fluor® 594 rabbit anti-goat IgG (H+L) or Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:100, Invitrogen).

Techniques: Staining, Control, Immunostaining, Two Tailed Test, Expressing, Knockdown, Binding Assay

Journal: Oncogene

Article Title: Arylsulfatase B Regulates Versican Expression by Galectin-3 and AP-1 Mediated Transcriptional Effects

doi: 10.1038/onc.2013.483

Figure Lengend Snippet: Panel A. To further assess the impact of ARSB silencing and galectin-3 silencing on versican expression, versican promoter activity was determined following ARSB silencing, galectin-3 silencing, and the combination of ARSB and galectin-3 silencing. In the prostate stromal cells, ARSB silencing increased versican promoter activity to 3.14 times the baseline (p<0.001). This increase was completely inhibited when both ARSB and galectin-3 were silenced. Negative vector control demonstrates no changes; positive actin control is not shown. No further decline in versican promoter activity occurred when ARSB and galectin-3 were both silenced and the inhibitors were present. Panel B. In the prostate epithelial cells, when ARSB was silenced, versican promoter activity increased to 2.70 times the baseline activity (p<0.001). This increase was completely abrogated by the combination of ARSB and galectin-3 silencing, demonstrating dependence of the ARSB knockdown-induced effect on the versican promoter on galectin-3, and consistent with the observed change in galectin-3 binding to C4S when ARSB was silenced. [ARSB=Arylsulfatase b; C4S=chondroitin-4-sulfate] Panel C. Chromatin immunoprecitiation (CHiP) assay was performed with c-Fos antibody following ARSB silencing and in the presence of c-Fos oligonucleotide binding inhibitor (I2). Epithelial and stromal cells showed significant increases in % DNA captured following ARSB silencing. Panel D. CHiP assay with galectin-3 antibody also demonstrated increase following ARSB silencing that was inhbited in the presence of I2. Panel E. Increased intensity of the chromatin bands is apparent following immunoprecipitation in the presence of c-Fos in the stromal and epithelial cells. Panel F. Similarly, immunoprecipitation with galectin-3 demonstrates increased band intensity following ARSB silencing. Panel G. c-Fos from the nuclear fraction of stromal cells bound with to the AP-1 binding sequence coated onto the wells of an ELISA plate. Anti-rabbit c-Fos and secondary goat-anti-rabbit-IgG-FITC were used to detect the c-Fos and were measured by fluorescence. Following ARSB silencing, the bound c-Fos was 2.25 and 2.30 times the baseline level in the stromal cells and in the epithelial cells, respectively. The I2 inhibitor completely blocked the binding. Panel H. Galectin-3 binding to the AP-1 oligonucleotide consensus sequence was also increased following ARSB silencing and inhibited by I2. When galectin-3 binding was detected by a mouse anti-galectin antibody and goat anti-mouse-IgG-Rhodamine Red ™ X, the binding was shown to have increased to 4.3 and 4.3 times the baseline, in the stromal and epithelial cells, respectively. Panel I. In the ARSB null mouse prostate tissue, the nuclear c-Fos and galectin-3 were both increased. C-Fos increased to 2.51 times the baseline and galectin-3 to 2.54 times the baseline (p=0.0002, p<0.0001, unpaired t-test, two-tailed). Panel J. Schematic diagram shows reduced binding of galectin-3 to more sulfated chondroitin-4-sulfate (C4S) that is present when ARSB is diminished. This is associated with increased nuclear galectin-3 and increased association of galectin-3 with c-Jun and c-Fos, leading to increased transcription of versican. [ARSB=arylsulfatase B; blue box = sulfate group at non-reducing end of C4S].

Article Snippet: Preparations were washed with PBS, blocked in 5% normal horse serum (KPL, Inc., Gaithersburg, MD) incubated overnight with goat polyclonal versican antibody (AF3054, R&D) or rabbit polyclonal galectin-3 antibody (sc-20157, SCBT) at 4°C, then washed and stained with Alex Fluor® 594 rabbit anti-goat IgG (H+L) or Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:100, Invitrogen).

Techniques: Expressing, Activity Assay, Plasmid Preparation, Control, Knockdown, Binding Assay, Immunoprecipitation, Sequencing, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test

Journal: Oncogene

Article Title: Arylsulfatase B Regulates Versican Expression by Galectin-3 and AP-1 Mediated Transcriptional Effects

doi: 10.1038/onc.2013.483

Figure Lengend Snippet: Panel A. The amount of C4S that co-immunoprecipitates with versican increased to ~2.5 times the baseline level in the prostate stromal and epithelial cells when ARSB was silenced (p<0.001). Panel B . In contrast, the EGFR that co-immunoprecipitated with versican declined to about 17% of the baseline value (p<0.001), with higher levels in the epithelial than the stromal cells. Panels C,D . The total EGFR was almost three times greater in both the stromal and epithelial cells following ARSB silencing (p<0.001), although less was associated with the versican. Panel E. The prostate epithelial cells were deprived of EGF, then challenged with exogenous EGF (10 ng/ml × 24 h, and the BrdU incorporation was detected. BrdU incorporation was significantly greater in the ARSB silenced cells than in the control or control silenced cells, increasing by 29% (p<0.001; n=6).

Article Snippet: Preparations were washed with PBS, blocked in 5% normal horse serum (KPL, Inc., Gaithersburg, MD) incubated overnight with goat polyclonal versican antibody (AF3054, R&D) or rabbit polyclonal galectin-3 antibody (sc-20157, SCBT) at 4°C, then washed and stained with Alex Fluor® 594 rabbit anti-goat IgG (H+L) or Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:100, Invitrogen).

Techniques: Immunoprecipitation, BrdU Incorporation Assay, Control

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Primer sequences, annealing temperatures, and cycles used for semiquantitative PCR

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican and CCL2 mRNA levels in monocytes from patients with systemic sclerosis and healthy controls . Quantitative PCR analysis. Relative mRNA expression levels were calculated as a ratio of mRNA levels of the genes of interest to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars in the graph denote the mean. Differences between the groups were analyzed by Mann-Whitney U test. SSc, systemic sclerosis.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican splice variants and mRNA levels in monocytes from systemic sclerosis patients and healthy controls . (A) Molecular structure of full-length versican (V0), which has numerous chondroitin sulfate (CS) chains attached to its glycosaminoglycan (GAG)-binding domain. (B) mRNA structures of versican splice variants (V0, V1, V2 and V3). Versican is composed of a hyaluronan binding region (HABR, green), GAG-binding domains (blue and yellow), and epidermal growth factor-like, lectin-like, and complement-regulatory-like domains (EELC, red). Individual mRNA components are shown in the same color as their corresponding protein structures. Arrows denote primers used to amplify each splice variant. (C) Levels of versican V0, V1, V2, and V3 mRNA in systemic sclerosis (SSc) and control monocytes, analyzed using semi-quantitative PCR. Relative mRNA levels were calculated as a ratio of the expression level of the gene of interest to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each bar in the graph denotes the mean. Differences between the two groups were analyzed by Mann-Whitney U test. NS, not significant.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Binding Assay, Variant Assay, Control, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican V0 and V1 mRNA levels in systemic sclerosis and control monocytes . Quantitative PCR analysis. Relative mRNA levels were calculated as a ratio of the level of the gene of interest to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each bar in the graph denotes the mean. Differences between the groups were analyzed by Mann-Whitney U test. SSc, systemic sclerosis.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Control, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Versican V0 and CCL2 protein levels in monocyte culture supernatants . Versican V0 and CCL2 protein levels in monocyte culture supernatants derived from systemic sclerosis (SSc) patients and healthy controls. (A) Representative immunoblot evaluating versican V0 protein expression. Monocyte culture supernatants were concentrated, truncated, and applied to immunoblots. (B) Versican V0 protein levels in monocytes from 14 patients with SSc and 11 healthy controls, semiquantitatively measured by densitometry. (C) CCL2 protein levels in monocytes from 16 patients with SSc and 13 healthy controls. CCL concentration in culture supernatants was measured by an ELISA. Each bar in the graph denotes the mean. Results from the two groups were compared by Mann-Whitney U test.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Derivative Assay, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Versican is upregulated in circulating monocytes in patients with systemic sclerosis and amplifies a CCL2-mediated pathogenic loop

doi: 10.1186/ar4251

Figure Lengend Snippet: Formation of CCL2 and versican complex in circulating monocytes . (A) CCL2 was incubated with plastic plates coated with chondroitin sulfate (CS) or vehicle. Bound CCL2 was recovered and subjected to immunoblots (lanes 2 and 3, respectively). Lane 1, untreated CCL2 as a positive control. A representative result from three experiments is shown. (B) Versican and CCL2 cellular localization in monocytes from an systemic sclerosis (SSc) patient, assessed by multi-color immunocytostaining: upper panel, CCL2 (green), versican (red), and their merged image; lower panel, goldin-97 (green), versican (red), and their merged image. Nuclei were counterstained with TO-PRO3 (blue). A representative result from three independent experiments is shown. Original magnification, ×600.

Article Snippet: The cells were fixed with acetone and incubated with goat anti-human versican polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with rabbit anti-human CCL2 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse anti-human goldin-97 mAb (clone CDF4; Invitrogen), followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Invitrogen).

Techniques: Incubation, Western Blot, Positive Control