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. 2013 Apr 11;8(4):e61306. doi: 10.1371/journal.pone.0061306

Figure 2. Ability to express CRH in Neuro2A cells transiently transfected with wild-type or mutant preproCRH construct.

Figure 2

A) CRH levels of expression detected by realtime quantitative PCR in not transfected (NT) or transfected cells (wt or p.Pro30Arg) at three different times: 24 h, 48 h and 72 h. Each bar represents the mean ± S.E.M. (n = 3) of mRNA levels normalized to the basal CRH expression in Neuro2A cells (NT values) and to a housekeeping control gene (b-Actin). * t = −3.676 and p = 0.020 compared with wt at 24 h; ** t = 5.274 and p = 0.002 compared with wt at 24 h. B) Densitometric analysis of CRH immunoreactive proteins in subcellular fractions of the Neuro2A cells. Each bar represents the mean ± S.E.M. (n = 3) and protein content is expressed in arbitrary units. C) Levels of secreted CRH protein measured by ELISA. The ability of cells to secrete the CRH hormone was evaluated by measuring the protein level in cultured media of cells transfected either with the wild-type or the mutant construct at 24 h or 48 h after the transfection. Each bar represents the mean ± S.E.M. (n = 2) and protein content is expressed as % in respect to the mean value of wt 24 h (assumed equal to 100%).* t = −7.403 and p = 0.005 compared with wt at 24 h; ** t = 7.796 and p = 0.004 compared with wt at 24 h.