Figure 9.
Effect of antisense A19 oligonucleotide on the polarity of plasma membrane proteins in CACO-2 cells C2BBe1. The cells were continuously grown in random (lanes A, B, E, F, I, and J; control) or antisense A19 (lanes C, D, G, H, K, and L) oligonucleotides. For these experiments, the cells were plated on 24-mm TranswellTM filters and cultured at confluency for 9 d. The monolayers were biotinylated from either the apical side (lanes A, C, E, G, I, K; Ap) or from the basolateral side (lanes B, D, F, H, J, L; Bl). Then, the filters were extracted in ice-cold PBS-EDTA supplemented with 2% Triton X-114. The supernatant of this extraction was warmed to 30°C for 3 min, and the detergent phase was acetone precipitated and run in SDS-PAGE (lanes I–L; TX-114). The pellets from the TX-114 extraction were then resuspended in PBS-EDTA, 1% Triton X-100 by sonication, and warmed up to 37°C for 15 min. The supernatants (lanes E–H; TX-100) and pellets (lanes A–D; Pellet) of this second extraction were also run in SDS-PAGE. In all cases the total amount of protein was measured to ensure that all lanes for a given extraction procedure were seeded with the same amounts of cellular material. All the lanes were blotted onto nitrocellulose sheets and probed with streptavidin–peroxidase and a chemiluminescence reaction. The small arrows between K and L point at apical bands now appearing also in the basolateral labeled set of proteins. The arrowheads indicate the position of molecular weight standards: (lanes A–D) 193, 112, 86, 70, 57, and 36 kD; (lanes E–L) 205, 116, 66, 45, and 29 kD. All the blots are from the same experiment, although, for technical reasons, they were run in separate gels with two different sets of molecular weight standards. Biotinylation control: CACO-2 C2BBe monolayers were grown on filters, incubated in A19, and biotinylated as described above. The cells were extensively washed, fixed in PFA, and processed with fluorescein-coupled streptavidin from both sides of the filter. Optical confocal sections were taken at the transnuclear plane (a) or at the apical membrane plane (b). Bars, 10 μm.