Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 1. Defined carboxy-terminal IG- FBP-2 fragments, but not intact IG- FBP-2, stimulate proliferation of growth plate chondrocytes in primary culture. Effect of intact IGFBP-2 (A), IGFBP-2104–289 (B), IGFBP-2167–289

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques:

Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 2. Defined carboxy-terminal IG- FBP-2 fragments, but not intact IG- FBP-2, stimulate proliferation of RCJ cells. Effect of intact IGFBP-2 (A), IG- FBP-2104–289 (B), IGFBP-2167–289 (C), and of IGFBP-2167–279 (D) alone (7.8 nM) and in coincubation with equimolar concentrations of IGF-I on DNA syn- thesis, as assessed by [3H]thymidine in- corporation assay. Subconfluent RCJ cells were cultured in serum-free me- dium for 24 h. Medium was changed to MEM containing 0.2% BSA, and pep- tides (dissolved in PBS) or vehicle were added as indicated. [3H]thymidine in- corporation was determined as de- scribed in Materials and Methods. Data are the mean SE expressed as percent of control. Statistics were by ANOVA of 24 parallel dishes per group. Experi- ments were performed at least three times. *, P 0.05 vs. control; #, P 0.05 vs. IGF-I.

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques: Cell Culture, Control

Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 4. Cell membrane binding of in- tact and fragmented IGFBP-2. A, The membrane (M) and cytosolic (C) frac- tions were isolated as described in Ma- terials and Methods. To verify cell com- partment separation, cell lysates from RCJ cells were run in parallel and probed for the cytoplasmic fraction with an antibody against GAPDH and for the plasma membrane preparation with an antibody against Na-K-AT- Pase. B, Western immunoblot analysis of the respective cell compartments in- cubated with vehicle or intact or frag- mented IGFBP-2 for 2 h. The respective cell lysates of RCJ cells were run in parallel and probed with a specific an- tibody against IGFBP-2, as described in the Materials and Methods.

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques: Membrane, Binding Assay, Isolation, Clinical Proteomics, Western Blot

Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 5. Intact IGFBP-2 and IGFBP-2104–289 bind to the cell surface. RCJ cells were plated on coverslips placed in 60-mm plates and treated with vehicle, intact IG- FBP-2 (7.8 nM), and IGFBP-2104–289 (7.8 nM) for 1 h, followed by washing with PBS and incubating again for 1 h with serum-free MEM. Cells were incubated with polyclonal rabbit anti-IGFBP-2 antibody, fixed, and in- cubated with secondary antibody as described in the Materials and Methods (magnification, 40). Strong im- munofluorescence was observed at the surface of RCJ cells incubated with intact IGFBP-2 and a labeled anti- IGFBP-2 antibody (A and B) and IGFBP-2104–289 (D and E). Some fluorescence was also present at the surface of the nucleus (staining in blue). No fluorescence was de- tectable in control cells incubated with vehicle and the anti-IGFBP-2 antibody (C and F).

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques: Incubation, Labeling, Fluorescence, Staining, Control

Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 6. A, Partially reduced full-length IG- FBP-2 is still able to bind IGF-II. Intact IGFBP-2 was incubated with varying con- centrations of DTT. Mild reducing condi- tions with a DTT concentration of 5.0 mM led to a partial reduction of IGFBP-2, which was still able to bind IGF-II, al- though to a lesser degree (66%) than un- treated (intact) full-length IGFBP-2. B, Partially reduced full-length IGFBP-2 does not contain proteolytic fragments of IG- FBP-2. Intact IGFBP-2 was incubated with varying concentrations of DTT, followed by immunoblot analysis using a polyclonal IG- FBP-2 antibody. C, Partially reduced full- length IGFBP-2 stimulates cell prolifera- tion to a comparable extent as equimolar IGF-I. RCJ cells were cultured until con- fluence, serum-starved for 12 h and stim- ulated with IGF-I (7.8 nM) and intact IG- FBP-2 (7.8 nM) incubated with or without DTT for an additional 48 h. Control cells were cultured without IGF-I in the absence or presence of DTT (5 mM for 30 min at 37 C). [3H]thymidine incorporation into the acid-extractable pool was determined by scintillation counting and used as a mea- sure of DNA synthesis, as described in Ma- terials and Methods. Data are mean SE. Statistics were by ANOVA were for 12 par- allel wells per group from three indepen- dent experiments. *, P 0.05 vs. control.

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques: Incubation, Concentration Assay, Western Blot, Cell Culture, Control, DNA Synthesis

Journal: Endocrinology

Article Title: Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I.

doi: 10.1210/en.2007-1395

Figure Lengend Snippet: FIG. 7. Both fragmented and partially reduced full-length IGFBP-2 stimulate the MAPK/ERK 1/2 and FAK signaling pathways. A, RCJ cells were serum starved for 12 h and incubated for 1 h with the respective IGFBP-2 peptides: control (lane 1), intact IGFBP-2 (lane 2), IGFBP-2104–289 (lane 3), IGFBP-2167–289 (lane 4), IGFBP-2167–279

Article Snippet: Polyclonal antihuman IGFBP-2 antibody (catalog no. PAB-IGFBP2–1) and intact human IGFBP-2 (catalog no. BP2BU020) were obtained from GroPep (Adelaide, Australia).

Techniques: Protein-Protein interactions, Incubation, Control