recombinant type 23 collagen ectodomain  (R&D Systems)

Journal: United European Gastroenterology Journal

Article Title: Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease

doi: 10.1177/2050640620977371

Figure Lengend Snippet: Specificity assessment of the PRO‐C23 antibody 10F6. (a) Sequence alignment for C‐terminus of type XIII, XXIII and XXV collagens. The antibody recognises the residues from 531 to 540 of type 23 collagen. (b) Western blot results of recombinant type 23 collagen using 10F6 as a primary antibody. (c) PRO‐C23 antibody specificity towards different peptides. Reactivity to the type XXIII collagen selection peptide (GLPVPGCWHK), the elongated peptide (GLPVPGCWHKA), the truncated peptide (GLPVPGCWH), a peptide from type XIII collagen (GLPVQGCWNK) and peptide from type XXV collagen (GLPMPGCWQK) was tested in the PRO‐C23 assay. (d) Correlations of PRO‐C23 levels serum levels with the levels of the EDTA, heparin and citrate plasma ( n = 16). PRO‐C23: biomarker of the ectodomain of type 23 collagen; OD: optical density; EDTA: ethylenediaminetetraacetic acid

Article Snippet: Western blot of recombinant type 23 collagen ectodomain (4165‐CL, R&D system) showed that the chosen antibody 10F6 recognised type 23 collagen ectodomain around 60 kD, while the reference commercial antibody (MAB4165, R&D system) was also shown (Figure ).

Techniques: Sequencing, Western Blot, Recombinant, Selection, Clinical Proteomics, Biomarker Discovery

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: COL23A1 , R & D Systems , 4165-CL , ECM.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: Molecular Biology of the Cell

Article Title: Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing

doi: 10.1091/mbc.E19-03-0140

Figure Lengend Snippet: Fibronectin matrix assembly is necessary for proteolytic processing of procollagen I. (A) GM03349 human dermal fibroblasts were grown to confluence as shown in the phase images (BF) in the presence of control peptide (III-11C) or FUD peptide to inhibit FN matrix assembly. Cells were fixed and costained for FN and collagen I (colI) with antibodies hFN7.1 and PA2140-2, respectively, followed by the appropriate fluorescent secondary antibodies. Representative epifluorescence images of a single field are shown for each peptide treatment. Scale bar: 10 μm. (B) Cells grown and treated as in A were lysed in either DOC or SDS buffer. DOC-insoluble material was collected from DOC lysates by centrifugation and then solubilized by boiling in buffered SDS. Samples were separated by electrophoresis in a 5% polyacrylamide gel and immunoblotted with anti-COL1A1 (top) or anti-FN (R184) antibody. Locations of procollagen, pN- or pC-collagen, and cleaved collagen I are indicated (right); two bands can be detected in the pNc/pCc band in lysates from subconfluent cells. Molecular mass standards are indicated on the left.

Article Snippet: Recombinant human BMP-1 (rhBMP-1) and recombinant human mini-procollagen (rhPro-COL1A1) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Centrifugation, Electrophoresis

Journal: Molecular Biology of the Cell

Article Title: Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing

doi: 10.1091/mbc.E19-03-0140

Figure Lengend Snippet: Procollagen colocalizes with FN fibrils. GM03349 cell cultures at confluence were coimmunostained for FN and either (A) pN collagen (pN) with anti–N-propeptide antibody or (B) collagen (col) with a polyclonal anti-COL1A1 antibody. Primary antibodies were detected with appropriate fluorescent secondary antibodies (FN, red; collagens, green) and confocal microscopy. Scale bar: 10 μm.

Article Snippet: Recombinant human BMP-1 (rhBMP-1) and recombinant human mini-procollagen (rhPro-COL1A1) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Confocal Microscopy

Journal: Molecular Biology of the Cell

Article Title: Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing

doi: 10.1091/mbc.E19-03-0140

Figure Lengend Snippet: Heparin enhances procollagen cleavage by rhBMP-1 in solution. GM03349-conditioned medium (A, B) or rhPro-COL1A1 (C, D) was mixed with rhBMP-1 ± heparin and incubated for the indicated times. Aliquots were removed from each reaction and stopped with a solution of SDS, DTT, and EDTA. Time-course samples from reactions supplemented only with rhBMP-1 (A, C) or with rhBMP-1 plus heparin (B, D) were immunoblotted with anti-procollagen N-propeptide antibodies. Untreated GM03349-conditioned media were electrophoresed in the first lanes (A, B). Locations of procollagen (pro), pN collagen (pNc), or mini-procollagen (mini-pNc) equivalents are indicated to the right of each blot. Molecular mass markers are to the left of each panel. Blots are representative of four experiments. See Supplemental Figure S5A for quantification.

Article Snippet: Recombinant human BMP-1 (rhBMP-1) and recombinant human mini-procollagen (rhPro-COL1A1) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Incubation

Journal: Molecular Biology of the Cell

Article Title: Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing

doi: 10.1091/mbc.E19-03-0140

Figure Lengend Snippet: Heparin enhances processing of procollagen by matrix-bound BMP-1. GM03349 cells plated at various densities (cells/cm 2 ) were grown for 72 h to yield increasing amounts of FN matrix indicated by the black triangle (see Supplemental Figure S7). Cultures were then incubated with rhBMP-1 (A) or rhBMP-1 + heparin (B) followed by rhPro-COL1A1 for 3 h. The rhPro-COL1A1 solution was collected, and equal aliquots were immunoblotted with an anti-procollagen N-propeptide antibody for observation of rhPro-COL1A1 processing. Locations of full-length mini-procollagen (mini-pro) and C-propeptide-cleaved/ N-propeptide-retaining mini-procollagen (mini-pNc) are indicated (right). Blots are representative of four experiments. See Supplemental Figure S5B for quantification.

Article Snippet: Recombinant human BMP-1 (rhBMP-1) and recombinant human mini-procollagen (rhPro-COL1A1) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Incubation

Journal: Molecular Biology of the Cell

Article Title: Fibronectin matrix as a scaffold for procollagen proteinase binding and collagen processing

doi: 10.1091/mbc.E19-03-0140

Figure Lengend Snippet: Model for FN-dependent proteolytic processing of procollagen I. Left, proteolytic cleavage of the C-terminal propeptide of procollagen I occurs when BMP-1 is bound to the FN matrix. An increased rate of processing is observed when both BMP-1 and heparin (or HS) are added together to the matrix. Right, example of procollagen and FN alignment to facilitate processing. Procollagen I contains a ¾ FN-binding site located ∼225 nm (or ¾ of the distance) from the N-terminus of a fully processed, 300-nm-long collagen molecule ( Dzamba et al. , 1993 ; Erat et al. , 2013 ). When this site is aligned with the collagen/gelatin-binding domain (GBD) on FN, the HepII domain of FN, located 55–60 nm from the N-terminus , is adjacent to the cleavage site of the C-propeptide of procollagen. Heparin binding to FN could facilitate procollagen cleavage by accumulating BMP-1 molecules at this site. Heparin/HS could also act as a scaffold for cleavage by simultaneously binding to BMP-1 molecules and to the C-terminal heparin-binding site on procollagen ( San Antonio et al. , 1994 ).

Article Snippet: Recombinant human BMP-1 (rhBMP-1) and recombinant human mini-procollagen (rhPro-COL1A1) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: COL23A1 expression in ccRCC tissues and paired ANTs. ( A ) Results of qRT-PCR exhibited the relative mRNA levels of COL23A1 in primary non-metastatic ccRCC tissues compared with paired ANTs from FUSCC cohort. Error bars are ± SD (n = 3). ( B ) The expression of COL23A1 mRNA in primary non-metastatic ccRCC tissues compared with paired ANTs from TCGA cohort. ( C ) Results of western blot analysis showed the COL23A1 protein levels in primary non-metastatic ccRCC tissues compared with paired ANTs from FUSCC cohort.

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: The correlation between COL23A1 expression and prognosis in ccRCC. ( A , B ) Representative high COL23A1 level in ccRCC tissues with ( A ) ×200 magnification and ( B ) ×400 magnification. ( C,D ) Representative low COL23A1 level in ccRCC tissues with ( C ) ×200 magnification and ( D ) ×400 magnification. ( E,F ) Representative COL23A1 level in normal kidney tissues with ( E ) ×200 magnification and ( F ) ×400 magnification. ( G ) Kaplan-Meier curves for OS after radical nephrectomy.

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: Clinicopathological characteristics in relation to COL23A1 expression status.

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of OS in 151 ccRCC patients.

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: The mRNA and protein expression of COL23A1 in normal and ccRCC cell lines. ( A ) A statistical analytical graph showed COL23A1 mRNA levels in the human kidney cortex cell lines (HKC) and four ccRCC cell lines (786-O, A-498, Caki-1, OS-RC-2). Error bars are ± SD (n = 3). ( B ) Results of western blot analysis showed COL23A1 protein levels in the human kidney cortex cell lines (HKC) and four ccRCC cell lines (786-O, A-498, Caki-1, OS-RC-2).

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: COL23A1 was down-regulated by small interfering RNA (siRNA) in ccRCC cell lines. ( A , B ) Relative COL23A1 mRNA levels in (A) 786-O and ( B ) A-498 cells that were transfected with siRNAs (#1, #2, #3) compared with siNC respectively were analyzed using qRT-qPCR. Error bars are ± SD (n = 3). ( C ) Results of western blot analysis showed the COL23A1 protein level of 786-O cells that were transfected with siRNAs (#1, #2, #3) compared with siNC, and the histogram right was quantification of the western blot analysis results. Error bars are ± SD (n = 3). ( D ) Results of western blot analysis showed the COL23A1 protein level of A-498 cells that were transfected with siRNAs (#1, #2, #3) compared with siNC, and the histogram right was quantification of the western blot analysis results. Error bars are ± SD (n = 3).

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Small Interfering RNA, Transfection, Western Blot

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: Effect of COL23A1 silencing on cell proliferation and cell cycle in ccRCC cells. ( A ) CCK-8 assays showed the effect of COL23A1 knockdown on the proliferation of 786-O cells. Error bars are ± SD (n = 3). ( B ) CCK-8 assays showed the effect of COL23A1 knockdown on the proliferation of A-498 cells. Error bars are ± SD (n = 3). ( C ) Effect of COL23A1 silencing on the cell cycle of 786-O cells. Error bars are ± SD (n = 3). ( D ) Effect of COL23A1 silencing on the cell cycle of A-498 cells. Error bars are ± SD (n = 3).

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: CCK-8 Assay, Knockdown

Journal: Scientific Reports

Article Title: The Oncogenic Role of COL23A1 in Clear Cell Renal Cell Carcinoma

doi: 10.1038/s41598-017-10134-2

Figure Lengend Snippet: Effect of COL23A1 silencing on cell migration and adhesion in ccRCC cells. (A, B) Effect of COL23A1 silencing on the cell adhesion of (A) 786-O and (B) A-498 cells. Error bars are ± SD (n = 3). (C, D) Effect of COL23A1 silencing on the expression of proteins involved in cell adhesion of (C) 786-O and (D) A-498. (E) Representative images of migration assays performed using 786-O (upper left) and A-498 (lower left) cells (scale bars, 100 µm). The histograms right were quantification of the migration assays of 786-O (upper) and A-498 (lower) cells. Error bars are ± SD (n = 3).

Article Snippet: Slides were blocked using normal goat serum followed by the avidin/biotin blocking kit and incubated with anti-COL23A1 antibody (1:200, Catalog # MAB4165, R&D Systems, Minneapolis, Minnesota, USA) at 4 °C overnight.

Techniques: Migration, Expressing