PMR60 over-expression decreases PTH mRNA and protein levels in transfected HEK293 cells. A. Northern blot analysis for rat (r) PTH or GH mRNA in HEK293 cells transiently co-transfected with plasmids for rat rPTH or GH and either catalytically active Myc-PMR60, or control (pSG5) plasmid at 48 h. Bottom panels: ethidium bromide staining of the membranes. Similar results were obtained in 4 independent experiments. B. Secreted rPTH in the medium of HEK293 cells in 3 repeat experiments as in A at 24 and 48 h. *, p < 0.05. C. Northern blot analysis for human (h) PTH mRNA in HEK293 cells transiently co-transfected with expression plasmids for hPTH and either PMR60 or a control plasmid (pSG5) at the indicated concentrations at 48 h. Quantification of PTH/L32 mRNA levels is shown below the gels. Similar results were obtained in 3 independent experiments. D. Secreted hPTH in the medium of HEK293 cells in 2 repeat experiments as in C. E. qRT PCR analysis for PTH and β-actin mRNA levels in HEK293 cells (n = 5) transfected with expression plasmids for rPTH or rPTH lacking the ARE and either catalytically active Myc-PMR60, or a control (pSG5) plasmid at 48 h. F-G. Effect of PMR60 over-expression on GH mRNA containing the PTH mRNA 63 nt ARE (GH63). F. Northern blot analysis of GH and control β-actin mRNA levels in cells transfected with plasmids for either GH or GH63 and PMR60 or control plasmids. G. Quantification of GH mRNA levels obtained in E and in an additional independent experiment, performed in triplicate. Results are shown as mean ± SE of mRNA in cells transfected with either GH or GH63 and pSG5 plasmid. *, p < 0.05.
Nechama et al. BMC Cell Biology 2009 10:70 doi:10.1186/1471-2121-10-70