Fig. 2

WT and mutated Kir4.1 expression and distribution in U251 astrocytoma cells.

(a) Co-immunofluorescence staining of Kir4.1 WT and R18Q expressing cells using Anti-Xpress epitope Ab to stain recombinant Kir4.1 (red) and phallacidin to stain actin filaments (green) shows that WT channels are mostly localized in the cytoplasm, and at plasma membrane in a low percentage of cells (top panels, arrows), while R18Q mutant channels are mainly distributed along cell membranes, filopodia-like structures and cell-cell contacts (bottom panels, arrowheads), and partially co-localizes with actin, in the majority of cells (70%). (b,c) Western Blot (WB) analysis of total membrane and cytosolic protein extracts of astrocytoma cells expressing WT and R18Q Kir4.1, probed with anti Kir4.1 Ab (b) and Anti-Xpress epitope tag Ab (c) revealed that R18Q is more abundantly expressed in the cytoplasm (CYT) and particularly in the total membrane protein fraction (MEM), than the WT protein. Anti-Xpress epitope tag Ab does not detect Kir4.1 protein in the cytoplasm. Arrowheads on the right of panel b highlight the monomeric and oligomeric forms of the Kir4.1 channel. Actin is used as loading control. Molecular weight markers are on the left (kDa). (d) Densitometric analysis of recombinant Kir4.1 bands derived from total membrane protein extracts from WT or R18Q Kir4.1 expressing cells, detected by anti-Xpress Ab and normalized to the corresponding actin value (mean ± SEM, expressed as arbitrary units; *P < 0.05 from three independent experiments). (e) WB of total cell proteins (SM) and enriched fraction of plasma membrane proteins after biotinylation experiments (Eluates) probed with anti-Kir4.1 Ab. Higher amount of R18Q Kir4.1 is expressed at the plasma membrane (arrow) when compared to Kir4.1 WT. Among Kir4.1 associated proteins α-syntrophin (α-synt) is found at plasma membrane of Kir4.1 R18Q mutant expressing cells (asterisk) but not in WT expressing cells. No differences are observed in β-dystroglycan (β-DG) and AQP4 association to astrocytoma plasma membrane. One representative experiment out of two performed with the same results has been shown.