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Fig. 5. Radiolabelling of kAE1 and kAE1(R901Stop) in polarised (A,E) or non-polarised (B,C,D) MDCKI cells. Cells were preincubated with sodium butyrate and labelled with 200 µCi/ml Easy-Tag^TM [³⁵S]methionine for 30 minutes (A,C,D) and pulse chased, or steady-state labelled for 4 hours (B,E). Immunoprecipitates were prepared using anti-AE1 Bric 155 (A) or Bric 170 (B-E) as described in Materials and Methods. Immunoprecipitates were treated with endo H or PNGase F where indicated and analysed by SDS-PAGE. (A) shows a pulse-chase experiment on MDCKI-kAE1 cells polarised on filters. The p3.1 vector control is shown in lane 1. (B) Steady-state labelled non-polarised MDCKI-kAE1 and MDCKI-kAE1(R901Stop) cells, respectively. (C,D) Pulse chase studies on non-polarised MDCKI-kAE1 cells (C) or MDCKI-kAE1(R901Stop) cells (D). (E) Selective biotinylation of the apical or basolateral surfaces of kAE1 and kAE1(R901Stop) cells polarised on filters. The apical and basolateral surface was selectively biotinylated, total AE1 immunoprecipitated and the biotinylated fraction selected using streptavidin beads. AP, apical; BL, basolateral.

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