recombinant type 23 collagen ectodomain  (R&D Systems)

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