Hypoxia (3%) and glucose deprivation induces regulator of calcineurin (Rcan)1-4 protein and mRNA expression in primary cortical murine astrocytes. (A, B) Immunoblots showing endogenous Rcan1-4, nuclear factor of activated T cells (NFAT)c1 and NFATc3 protein expression, with α-tubulin and PSF (PTB-associated splicing factor) expression detected as loading controls. (A) Rat primary cortical astrocytes were non-stimulated (ns), treated with phorbol ester (phorbol 12-myristate 13-acetate) plus A23187 calcium ionophore (PIo) (4 h) as a positive control, or subjected to combined hypoxia (3% O2) and glucose deprivation (HGD) for 1 to 6 h. (B) Mouse primary astrocyte cultures were non-stimulated (ns) or subjected to HGD for 30 minutes to 4 h. (C)
Rcan 1-4 mRNA was quantified by TaqMan real time qRT-PCR on total RNA from primary cortical mouse astrocytes stimulated as in (B). Transcript amounts are normalized to 18S rRNA and TATA-binding protein (TBP) endogenous controls, and are expressed relative to the level in non-stimulated control cells (ns). Data are the means ± SD of triplicate real time qRT-PCR determinations for each condition; n = 4. **P < 0.01 (analysis of variance (ANOVA)) versus ns.