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. Author manuscript; available in PMC: 2013 Sep 19.
Published in final edited form as: J Endocrinol. 2009 Dec 23;204(3):287–297. doi: 10.1677/JOE-09-0183

Figure 4. Knockdown of CaR mRNA expression inhibits the effects of high calcium and neomycin on cAMP, PTHrP and ACTH.

Figure 4

A) Measurement of CaR mRNA levels by quantitative, real-time RT-PCR in AtT-20 cells transfected with two different, specific siRNA (CaR-siRNA-652 and CaR-siRNA-653) against the CaR as well as with a control, non-specific siRNA. CaR-specific siRNAs caused approximately a 75% reduction in CaR mRNA levels as compared with the untransfected cells. Bars represent the mean ± S.E. of three individual experiments. * denotes significant differences between CaR-siRNAs (652 and 653) versus untransfected cells (p < 0.001). B), C) & D) show the effects of these CaR siRNAs on intracellular cAMP and PTHrP and ACTH secretion, respectively, in response to CaR activation by high calcium (5 mM) and neomycin. CaR activation failed to increase cAMP, PTHrP or ACTH levels in cells transfected with specific CaR-siRNAs (652 and 653). However, non-transfected cells and cells transfected with non-specific CaR-siRNA (siRNA-control) demonstrated the expected stimulation of cAMP, ACTH and PTHrP levels in response to 5 mM calcium or neomycin. Bars represent the mean ± S.E. of three independent experiments. * denotes significant differences between CaR-siRNAs (652 and 653) versus non-transfected cells, (p < 0.001).