Fig. 5

The hZip1-myc protein was not detected at the plasma membrane of Caco-2 cells, and hZip1-myc-tagged protein was not internalised by the cells. A Western blot analysis of the Caco-2 cell extracts probed with anti-myc antibody, with β-actin expression as loading control, representative of 3 independent experiments. Lane 1, Caco-2 cells transfected with pcDNA3 vector control; Lane 2, Caco-2 cells transfected with hZip1-myc construct, showing 35 kDa band of expected size. B immunolocalisation of hZip1 protein in Caco-2 cell line. Permeabilised (a, c) and non-permeabilised (b, d) cells were labelled with anti-myc antibody. No staining in vector control Caco-2 cells (a and b). No label in non-permeabilised Caco-2 (d) cells. Permeabilised Caco-2 cells had a granular cytoplasmic pattern (c). C Live cells were incubated with 5 μg/ml anti-myc or anti-transferrin receptor antibodies for 1 h. The hZip1-myc Caco-2 cells were either maintained in growth media (a, c) or in media supplemented with 6 μM TPEN (b, d). Surface binding or internalisation of anti-myc antibody was not detected in cells (a, b) while anti-transferrin receptor antibody was found at the plasma membrane and in the cytoplasm (c,)