Figure 1

Over-expression of the CaR decreases PTH mRNA levels in co-transfected HEK293 cells. HEK293 cells were transiently co-transfected in triplicate with expression plasmids for the hPTH gene and control GFP or GH and either the CaR (CaR+) or empty vector (CaR-). A. Schematic representation of the human PTH expression plasmid used for transient transfections. The boxes show the CMV promoter (grey) and the PTH exons with the untranslated regions (UTRs) (white) and coding regions (diagonal lines). The arrows show the pre, pro and mature PTH. B. Immunohistochemistry on whole cells using an intact cell enzyme-linked immunoassay for the cell surface expression of the CaR. Untransfected (background), CaR (+) or control plasmid (-) transfected cells were analyzed using a CaR antibody or IgG (-). C. Immunoblot analysis of extracts from HEK293 cells co-transfected with expression plasmids for the CaR and myc-AUF1 as control plasmid, using anti-CaR or myc antibodies. D-G. Effect of CaR on PTH expression. D. Northern blot for hPTH and co-transfected GFP with the CaR (+) or an empty vector (-). Ethidium bromide staining of the membrane is shown as a loading control. E. qPCR for PTH and co-transfected GH mRNA levels from cells with (red) and without (blue) the CaR. F. Secreted PTH from cells as above 1 h after an incubation in fresh medium, 1 mM Ca²⁺. G. qRT-PCR for PTH mRNA levels from cells without and with either the CaR or the PTH1R (checkered). Data in D-G are expressed as fold change (mean ± SE) (n = 3). *, P < 0.01, CaR: control.