Cyclosporin A (CsA) pretreatment and regulator of calcineurin (Rcan)1-4 overexpression cause the appearance of slower migrating nuclear factor of activated T cells (NFAT)c3 forms on SDS-PAGE. (A, B) Total cell lysates were separated on 6% SDS-PAGE gels and immunoblotted with anti-NFATc3 antibody. Ponceau stainings of membranes are presented as loading controls. (A) Mouse primary astrocyte cultures were either left without pretreatment or were pretreated with CsA for 1 h as indicated. Cells then were left non-stimulated (ns) or were subjected to hypoxia and glucose deprivation (HGD) for 30 minutes to 4 h. (B) Three independent mouse astrocyte cultures (A-C) were infected with adenovirus bicistronically encoding green fluorescent protein (GFP) plus Rcan1-4 (Ad Rcan1-4) or encoding GFP alone (Ad GFP). Samples were run in parallel on 10% gels to analyze Rcan1-4 expression and to monitor GFP expression as a control of infection. Immunoblots show the effect of Ad Rcan1-4 infection on Rcan1-4 protein expression and NFATc3 electrophoretic mobility.