Fig. 3. The majority of N-terminal truncations of kAE1 are localised to the TGN. Nonpolarised MDCKI cells stably expressing Nt1-360AE1 (a), Nt1-338AE1 (b), Nt1-292AE1 (c), Nt1-269AE1 (d) or Nt1-155AE1 (e) or transiently expressing Nt1-338AE1(Y359A) (f), Nt1-244AE1 (g) and Nt1-214AE1 (h) were fixed and immunostained with Bric170 and a suitable secondary antibody. Only the Nt1-155AE1 truncation was correctly localised to the plasma membrane, whereas the other mutants were localised to the TGN. Merged image in k shows the overlap between Nt1-338AE1 detected with Bric170 (i) and TGN38 detected with a rabbit anti-TGN38 antibody (j). Mutation of Y359 to alanine did not alleviate the TGN trafficking of the Nt1-338AE1 protein when transiently expressed in MDCKI cells (compare f with b). Therefore inclusion of even small sections of the N-terminus to the Nt1-360AE1 protein appears to disrupt the steady state localisation of kAE1. Scale bar: 30 µm.