RCAN1 Regulates Mitochondrial Function and Increases Susceptibility to Oxidative Stress in Mammalian Cells
Figure 4
Generation of a stable PC12 cell line overexpressing RCAN1, . (a) Diagrammatic representation of the generation of the FLAG-fused RCAN1 construct, which was cloned into pGEM-T easy vector and finally ligated to the EcoR1 restriction site of pcDNA3 expression vector containing a pCMV promoter. (b) Agarose gel electrophoresis of the PCR products from the 3 selected clones containing the 900 bp RCAN1-FLAG tag sequence, a water only (−) control, and (+) control using DNA encoding the pGEM-T/RCAN1-FLAG construct. (c) Immunoblot against the FLAG tag in the 3 selected clones and a negative control lacking FLAG normalized to β-tubulin loading control. (d) Immunocytochemistry of control PC12 and cells with an antibody against RCAN1 demonstrates the elevated RCAN1 expression in cells (scale bar = 50 μm).
