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Figure 3. Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT (A) or K346T (B) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. (C) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. (D) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). (E) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, *P < 0.05). (F) The resting membrane potential and (G) current density (at −100 mV) were evaluated in cells expressing WT (white bars) or K346T (gray bars) channels (data are mean ± SEM; n = 6; *P < 0.05; **P < 0.01). Image published in: Ambrosini E et al. (2014) © The Author 2014. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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