Figure 4

GCs induce RCAN1-1 transcript and protein levels in CEM-C7-14 Cells in a kinase-dependent fashion: Panel A: CEM-C7-14 cells were treated for 24 h with the indicated kinase inhibitors in the presence of ethanol (E) or 100 nM Dex (D), total RNA was extracted, and analyzed by reverse transcription-Real-time Q-PCR using primers specific for RCAN1-1. Relative transcript expression in various conditions was calculated by the Pfaffl method using β-actin as a reference. Panel B: CEM-C7-14 cells were treated with the indicated kinase inhibitors in the presence of either ethanol (E) or 100 nM Dex (D) for 24 h prior to Western blotting with either RCAN1 or β-actin specific antibodies as in Panel A. Densitometric analysis of two independent experiments was performed using ImageJ software, and average data are presented below the gel. Panel C: CEM-C7-14 cells were treated with the indicated kinase inhibitors in the presence of either ethanol (E) or 100 nM Dex (D) for 24 h were subjected to (i) Western blotting with phospho-Akt specific antibody (Cell Signaling Technology, cat # 9271) or (ii) p38 MAP kinase activity using a kit from Cell Signaling Technology (cat # 9820), as described in the methods section.