Exendin 4

PDGF-BB was purchased form R&D System, Inc. (Minneapolis, MN, USA). GLP-1 and GIP were obtained from Peptide Institute, Inc. (Osaka, Japan). 8-Bromo cAMP, PD98059, SP600125 or SB203580 were obtained from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). Dibutyryl (Bt2) cAMP was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). H89 was obtained from Seikagaku Co. (Tokyo, Japan). Phospho-specific cAMP response element-binding protein (CREB) antibodies and phospho-specific p38 MAP kinase antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies, anti-osteocalcin mouse antibody and anti-Rho-A (26C4) mouse monoclonal antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfront, UK). Exendin-4 was obtained from Abcam (Cambridge, UK). Other materials and chemicals were obtained from commercial sources. H89, PD98059, SP600125 or SB203580 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.3%, which did not affect the cell migration assay or detection of the protein level using Western blotting^{22 (link)}.

The concentration of exendin-4 secreted by recombinant L. paracasei was measured by indirect ELISA according to the method described by Zhang et al. [41 (link)] with some modifications. Standard exendin-4 (Sigma, 0.2 μg/ml) was used as the coated antigen. Equal volumes (100 μl) of monoclonal antibody against exendin-4 (Abcam) (1:4000) and standard exendin-4 (0–100 nmol/l) or culture supernatant samples of L. paracasei L14/pMG76e-exendin-4, preincubated overnight at 4°C, were applied to detect the captured exendin-4. Horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam) (100 μl) was used at a 1:10,000 dilution and revealed by reaction with TMBS substrate (Amresco). Absorption was determined at 450 nm and 630 nm by Multiskan MK3 ELISA plate reader (Thermo Labsystems). The exendin-4 concentration was calculated by the standard curve method.