Regulator of calcineurin (Rcan)1-4 protein downregulates expression of the proinflammatory gene
in mouse primary astrocytes. (A) Astrocytes were infected with adenovirus bicistronically encoding green fluorescent protein (GFP) plus Rcan1-4 (Ad Rcan1-4) or encoding GFP alone (Ad GFP). Immunoblots show the effect of Ad Rcan1-4 infection on Rcan1-4 protein expression and nuclear factor of activated T cells (NFAT)c3 electrophoretic mobility. α-Tubulin and PSF (PTB-associated splicing factor) were used as endogenous loading controls and GFP as a marker of equal infection. (B) Rcan1-4 overexpression inhibits astrocyte cyclo-oxygenase 2 (Cox-2) induction. At 48 h post infection, cells were quiesced, and then stimulated with phorbol ester (phorbol 12-myristate 13-acetate) plus A23187 calcium ionophore (PIo) as indicated. Blots show expression of Cox-2 and Rcan1-4 protein. Identical concentrations of adenoviral particles were used for each infection in all experiments. (C) Densitometry analysis of PIo-Cox2 induction. Data are the means ± SD of densitometry value of n = 4 experiments. The figure of 100% is the Cox2 expression achieved by PIo stimulation for 6 h in Ad GFP cells. PIo-induced Cox2 expression in Ad Rcan1-4 cells is 72 ± 11%. All values are corrected relative to the loading control α-tubulin.