Journal: Endocrinology

Article Title: Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

doi: 10.1210/en.2003-0036

Figure Lengend Snippet: FIG. 1. Western blot evidence for the presence of LH receptors as- sociated with osteoblasts. In each instance a total of 15 g of total protein extract was loaded per lane and subjected to SDS-PAGE under reducing conditions. Separated proteins were subsequently transferred onto nitrocellulose and the membrane probed using an antibody that recognizes residues 1–11 (after the signal peptide) of the LH receptor exodomain. Lane 1 was loaded with an extract from the human osteoblast-like cell line MG63; lane 2 represents a primary hOB preparation, whereas lane 3 was loaded with proteins recovered from a murine osteoblast-like cell line mC3T3. Lanes 4 and 5 are positive controls and represent a murine Leydig cell (mLTC-1) and whole rodent ovary extract, respectively. Detection of the LH receptor was achieved using an alkaline phosphatase conjugated goat anti- rabbit antibody and a substrate combination of 3-bromo-4-chloro-5- indolylphosphate and nitroblue tetrazolium. All extracts exhibit a protein doublet between 66 and 68 kDa, in addition human and rat extracts are the only samples that exhibit the strongest staining for 105- and 135-kDa proteins. The rodent ovary extract also has a fur- ther component that runs at approximately 75 kDa.

Article Snippet: The resultant homogenate was then extracted over a period of 1 h under refrigerated conditions, the sample was centrifuged for 10 min at 2000 g, and the supernatant removed and stored at 20 C until required. hOBs, the human osteoblast-like cell line MG63, murine mC3T3-E1 osteoblast-like cells, and murine Leydig tumor cells (mLTC-1; ATCC, Manassas, VA) were grown under conventional culturing conditions in media recommended for each cell type.

Techniques: Western Blot, SDS Page, Membrane, Staining

Journal: Endocrinology

Article Title: Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

doi: 10.1210/en.2003-0036

Figure Lengend Snippet: FIG. 5. Treatment of osteoblasts (hOBs) with hCG to ascertain whether cAMP is generated. Both hOBs and the murine Leydig cell line, mLTC-1 (positive con- trol) were treated with 1 105 IU/liter hCG or vehicle for 10 min or 2 or 8 h. Following stimulation the culture dishes were treated to extract total cAMP for quantification by RIA. The data obtained indicate that hOBs are unable to gener- ate cAMP. In each case, a total of eight samples were processed for cAMP quan- tification, and the data were expressed as the mean nanomolar concentration of cAMP SEM.

Article Snippet: The resultant homogenate was then extracted over a period of 1 h under refrigerated conditions, the sample was centrifuged for 10 min at 2000 g, and the supernatant removed and stored at 20 C until required. hOBs, the human osteoblast-like cell line MG63, murine mC3T3-E1 osteoblast-like cells, and murine Leydig tumor cells (mLTC-1; ATCC, Manassas, VA) were grown under conventional culturing conditions in media recommended for each cell type.

Techniques: Generated, Concentration Assay