Journal: Biochimica et biophysica acta

Article Title: Human IGF-I Propeptide A Promotes Articular Chondrocyte Biosynthesis and Employs Glycosylation-dependent Heparin Binding

doi: 10.1016/j.bbagen.2017.11.017

Figure Lengend Snippet: Production and characterization of six human IGF-I prepropeptides in transfected bovine chondrocytes. Bovine chondrocytes were transfected by each of pAAV-IGF-I1A, 1B, 1C, 2A, 2B and 2C. The respective human IGF-I products were assessed in day 2 conditioned medium (CM) by (A) human IGF-I ELISA, (B) functional ELISA using heparin in place of the anti-IGF-I capture antibody in the human IGF-I ELISA and (C) western blotting of CM samples probed with ant-IGF-I antibody, using recombinant IGF-I (rIGF-I) for comparison. ELISA data are presented as mean ± SD of three independent experiments. Western blot data are from a representative experiment and equal amounts (20 ul) of CM were loaded for each sample. Human recombinant IGF-I (rIGF-I, 2 ng) was loaded for comparison. The western blot shows that the cells transfected with pAAV-IGF-I1A or 2A, produced mature IGF-I and two forms of IGF-I propeptide A (proIGF-IA). The cells transfected with pAAV-IGF-I1B or 2B, produced mature IGF-I and one IGF-I propeptide B (proIGF-IB). The cells transfected with pAAV-IGF-I1C or 2C, produced mature IGF-I and IGF-I propeptide C (proIGF-IC). *: p < 0.05; **: p < 0.01; ***: p < 0.001. Detailed p values are provided in STable 1.

Article Snippet: IGF-I in conditioned medium was measured by human IGF-I ELISA (R&D Systems, Cat.#: D291) according to the manufacturer’s procedures.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Functional Assay, Western Blot, Recombinant, Comparison, Produced

Journal: Biochimica et biophysica acta

Article Title: Human IGF-I Propeptide A Promotes Articular Chondrocyte Biosynthesis and Employs Glycosylation-dependent Heparin Binding

doi: 10.1016/j.bbagen.2017.11.017

Figure Lengend Snippet: Production and characterization of human IGF-I prepropeptide 1A truncated mutants. Bovine chondrocytes were transfected by each of pAAV-IGF-I1A-noEA and pAAV-IGF-I1A-noEx6 to express human IGF-I prepropeptide 1A without EA peptide (1A-NoEA) and human IGF-I prepropeptide 1A without the C-terminal 19 amino acid sequence of the Exon 6 (1A-noEx6). pAAV-IGF-IA transfection was included for comparison. The respective human IGF-I products were assessed in day 2 conditioned medium (CM) by (A) human IGF-I ELISA, (B) functional ELISA using heparin in place of the anti-IGF-I capture antibody in the human IGF-I ELISA and (C) western blotting of CM samples probed with ant-IGF-I antibody. ELISA data are presented as mean ± SD of three independent experiments. Western blot data are from a representative experiment and equal amounts (20 ul) of CM were loaded for each sample. The western blot shows that the cells transfected with pAAV-IGF-I1A-noEA (1A-NoEA) produced only mature IGF-I. The cells transfected with pAAV-IGF-I1A-noEx6 (1A-noEx6) produced mature IGF-I and the IGF-I containing the 16 AA sequence of Exon 4. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Detailed p values are provided in STable 2.

Article Snippet: IGF-I in conditioned medium was measured by human IGF-I ELISA (R&D Systems, Cat.#: D291) according to the manufacturer’s procedures.

Techniques: Transfection, Sequencing, Comparison, Enzyme-linked Immunosorbent Assay, Functional Assay, Western Blot, Produced

Journal: Biochimica et biophysica acta

Article Title: Human IGF-I Propeptide A Promotes Articular Chondrocyte Biosynthesis and Employs Glycosylation-dependent Heparin Binding

doi: 10.1016/j.bbagen.2017.11.017

Figure Lengend Snippet: Production and characterization of a human IGF-I prepropeptide 1A mutant (1A-N92A), in which the putative N-glycosylation site Asn92 in EA peptide was replaced by Ala. Bovine chondrocytes were transfected by pCMV-IGF-I1A-N92A. pAAV-IGF-I1A transfection and pAAV-IGF-I1A-noEA transfection were included for comparison. The respective human IGF-I products were assessed in day 2 conditioned medium (CM) by (A) human IGF-I ELISA, (B) functional ELISA using heparin in place of the anti-IGF-I capture antibody in the human IGF-I ELISA and (C) western blotting of CM samples probed with ant-IGF-I antibody. ELISA data are presented as mean ± SD of three independent experiments. Western blot data are from a representative experiment and equal amounts (20 ul) of CM were loaded for each sample. The western blot showed that the cells transfected with pCMV-IGF-I1A-N92A produced mature IGF-I and one IGF-I propeptide A (proIGF-IA) without N-glycosylation at Asn92 in the EA peptide. The cells transfected with pAAV-IGF-I1A produced mature IGF-I and two IGF-I propeptide A forms (proIGF-IA), one without N-glycosylation at Asn92 in the EA peptide and one with N-glycosylation at Asn92 in the EA peptide. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Detailed p values are provided in STable 3.

Article Snippet: IGF-I in conditioned medium was measured by human IGF-I ELISA (R&D Systems, Cat.#: D291) according to the manufacturer’s procedures.

Techniques: Mutagenesis, Glycoproteomics, Transfection, Comparison, Enzyme-linked Immunosorbent Assay, Functional Assay, Western Blot, Produced

Journal: Biochimica et biophysica acta

Article Title: Human IGF-I Propeptide A Promotes Articular Chondrocyte Biosynthesis and Employs Glycosylation-dependent Heparin Binding

doi: 10.1016/j.bbagen.2017.11.017

Figure Lengend Snippet: Comparison of proIGF-IA, BMP-2 and TGF-β1 heparin binding activity. CM from chondrocytes transfected with pAAV-IGF-I1A or PBS containing recombinant BMP-2 (10 ug) or TGF-β1 (8 ug), was analyzed by FPLC using a heparin-sepharose column. CM was applied to the column and the column was washed with 10 ml PBS. The column was eluted with a 10-ml linear gradient of sodium chloride from 0 to 2 M in PBS. The eluate was collected in 0.5 ml fractions. IGF-I, BMP-2 and TGF-β1 in eluate fractions were measured by human IGF-I ELISA, human BMP-2 ELISA and human TGF-β1 ELISA, respectively. Data are expressed as ng/ml of the designated growth factors in each fraction of the eluate.

Article Snippet: IGF-I in conditioned medium was measured by human IGF-I ELISA (R&D Systems, Cat.#: D291) according to the manufacturer’s procedures.

Techniques: Comparison, Binding Assay, Activity Assay, Transfection, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta

Article Title: Human IGF-I Propeptide A Promotes Articular Chondrocyte Biosynthesis and Employs Glycosylation-dependent Heparin Binding

doi: 10.1016/j.bbagen.2017.11.017

Figure Lengend Snippet: Production and characterization of two human IGF-I prepropeptide 1A mutants, in which the three Arg (Arg71, Arg74 and Arg77) in the EA peptide were replaced by Ala (1A-R/A) or Lys (1A-R/K). Bovine chondrocytes were transfected by pCMV-IGF-I1A-R/A or pCMV-IGF-I1A-R/K. pAAV-IGF-I1A transfection and pAAV-IGF-I1A-noEA transfection were included for comparison. The respective human IGF-I products were assessed in day 2 conditioned medium (CM) by (A) human IGF-I ELISA and (B) western blotting of CM samples probed with ant-IGF-I antibody. ELISA data are presented as mean ± SD of three independent experiments. Western blot data are from a representative experiment and equal amounts (20 ul) of conditioned medium were loaded for each sample. The western blot showed that the cells transfected with each of pCMV-IGF-I1A-R/A and pCMV-IGF-I1A-R/K, produced one major proIGF-IA with N-glycosylation at Asn92 in the EA peptide and one minor proIGF-IA without N-glycosylation at Asn92 in the EA peptide, but did not produce mature IGF-I. Additional transfected cells were cultured for 6 days and assayed for DNA content (C) and glycosaminoglycan (GAG) production (D). GAG released into the medium (Medium) and GAG retained with the chondrocytes in the cell layer (Cell layer) were measured separately. Chondrocytes transfected by empty vector were used as control. Data are normalized to control and presented as mean ± SD of three independent experiments. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Detailed p values are provided in STable 4.

Article Snippet: IGF-I in conditioned medium was measured by human IGF-I ELISA (R&D Systems, Cat.#: D291) according to the manufacturer’s procedures.

Techniques: Transfection, Comparison, Enzyme-linked Immunosorbent Assay, Western Blot, Produced, Glycoproteomics, Cell Culture, Plasmid Preparation, Control