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Fig. 3. Decreased GLT-1 level on the cell surface of G93A-expressing MDCK cell lines. (A) Biotinylation of surface GLT-1 in parental MDCK cells (M) and MDCK cells expressing wild-type SOD1 (S) or the G93A mutant (G). Forty-eight hours after GLT-1 transfection, the cells were pre-incubated with 5 mM EDTA to open the tight junctions and allow biotinylation of lateral surface proteins with sulfo-NSH-biotin; the surface biotinylated proteins were recovered on streptavidin beads. Equal volumes of total cell lysates (Tot), biotinylated surface proteins (Biot) and intracellular proteins (Ic) were separated by 10% SDS-PAGE and immunostained for GLT-1. The biotinylated proteins were also probed for E-cadherin (E-CAD) as surface markers. (B-C) Densitometric analysis of the expression of GLT-1 in M, S and G cell lines. The pixel densities of lanes from three separate experiments (one of which is shown in A) were evaluated using the NIH Image program. All of the values were expressed as the percentage of surface transporter compared to total expression of GLT-1 in each cell line±s.e.m. Statistical an alysis (t-test) revealed a highly significant reduction in surface GLT-1 in the G93A-expressing cell line (P=0.0063). (C) The pixel densities of lanes from the experiments shown in A (Tot, Biot) were evaluated using the NIH Image program and expressed as OD units.
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