Figure 3.
Figure 3. Overexpression of MyoVb tail blocks FcRn-dependent IgG transcytosis but not recycling. All experiments were performed in MDCK cells stably expressing a tetracycline-inducible EGFP–MyoVb tail and FcRn-HA. (a) Doxycycline induces expression of EGFP–MyoVb tail in nonpolarized (lanes 1 and 3) and polarized cells (lanes 2 and 4), as measured by SDS-PAGE and immunoblotting using monoclonal antibodies against EGFP. The bottom shows corresponding actin immunoblotting. **, cross-reacting protein band insensitive to doxycycline treatment. Numbers to the left indicate kD. (b) Doxycycline-induced expression of EGFP–MyoVb tail collapses Rab11-containing endosomes into a dense apical structure consistent with the ARE. The induced structure does not contain Tf-R. Polarized cells treated with doxycycline were fixed and immunostained for Rab11 and Tf-R. Bar, 10 µm. (c) Doxycycline-induced expression of EGFP–MyoVb tail inhibits transcytosis of NIP-IgG (shaded bars) in both directions as measured by ELISA; results are mean ± SD (n = 5). NIP-IgG-IHH (open bars) was used as control for nonspecific transport. *, P < 0.001. (d) Doxycycline-induced expression of EGFP–MyoVb tail has no detectable effect on recycling of NIP-IgG (shaded bars) to either apical or basolateral cell surfaces as measured by ELISA; results are mean ± SD (n = 3). NIP-IgG-IHH (open bars) is used as a control for nonspecific transport. (e) Doxycycline-induced expression of EGFP–MyoVb tail has no detectable effect on endocytosis of Nip-IgG (shaded bars) from either apical or basolateral membranes. Cells kept on ice (time 0) or incubated with NIP-IgG-IHH (open bars) provide control for nonspecific transport. A representative study with three independent measurements for each point is shown. Error bars indicate the SD of three independent experiments. (f) Doxycycline-induced expression of EGFP–MyoVb tail does not divert FcRn to the lysosomal compartment. Polarized cells treated or not treated with doxycycline were fixed and immunostained for FcRn (HA staining) and LAMP1. The distribution of colocalization of FcRn with LAMP1 was quantified using the Manders coefficient as described (see main text and Materials and methods). Horizontal bars in the graphs indicate that mean Mtarget protein equals 0.04 and 0.08 for +Dox and −Dox, respectively.

Overexpression of MyoVb tail blocks FcRn-dependent IgG transcytosis but not recycling. All experiments were performed in MDCK cells stably expressing a tetracycline-inducible EGFP–MyoVb tail and FcRn-HA. (a) Doxycycline induces expression of EGFP–MyoVb tail in nonpolarized (lanes 1 and 3) and polarized cells (lanes 2 and 4), as measured by SDS-PAGE and immunoblotting using monoclonal antibodies against EGFP. The bottom shows corresponding actin immunoblotting. **, cross-reacting protein band insensitive to doxycycline treatment. Numbers to the left indicate kD. (b) Doxycycline-induced expression of EGFP–MyoVb tail collapses Rab11-containing endosomes into a dense apical structure consistent with the ARE. The induced structure does not contain Tf-R. Polarized cells treated with doxycycline were fixed and immunostained for Rab11 and Tf-R. Bar, 10 µm. (c) Doxycycline-induced expression of EGFP–MyoVb tail inhibits transcytosis of NIP-IgG (shaded bars) in both directions as measured by ELISA; results are mean ± SD (n = 5). NIP-IgG-IHH (open bars) was used as control for nonspecific transport. *, P < 0.001. (d) Doxycycline-induced expression of EGFP–MyoVb tail has no detectable effect on recycling of NIP-IgG (shaded bars) to either apical or basolateral cell surfaces as measured by ELISA; results are mean ± SD (n = 3). NIP-IgG-IHH (open bars) is used as a control for nonspecific transport. (e) Doxycycline-induced expression of EGFP–MyoVb tail has no detectable effect on endocytosis of Nip-IgG (shaded bars) from either apical or basolateral membranes. Cells kept on ice (time 0) or incubated with NIP-IgG-IHH (open bars) provide control for nonspecific transport. A representative study with three independent measurements for each point is shown. Error bars indicate the SD of three independent experiments. (f) Doxycycline-induced expression of EGFP–MyoVb tail does not divert FcRn to the lysosomal compartment. Polarized cells treated or not treated with doxycycline were fixed and immunostained for FcRn (HA staining) and LAMP1. The distribution of colocalization of FcRn with LAMP1 was quantified using the Manders coefficient as described (see main text and Materials and methods). Horizontal bars in the graphs indicate that mean Mtarget protein equals 0.04 and 0.08 for +Dox and −Dox, respectively.

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