Rab25 regulates a sorting step that specifies transcytosis, whereas Rab11a regulates recycling in the basolateral pathway only. All experiments use doxycycline-induced shRNA against Rab25 (left panels) or Rab11a (right panels) in polarized FcRn-EGFP cells. (a) shRNA against Rab25 suppresses gene expression as assessed by RT-PCR. (b) shRNA against Rab11a suppresses gene expression as assessed by SDS-PAGE and immunoblotting. Numbers on the left indicate kD. (c and d) Gene suppression of Rab25 (c) or Rab11a (d) does not alter trafficking of FcRn–IgG complexes into the common RE. Alexa Fluor 568–IgG was applied apically and Alexa Fluor 647–Tf was applied basolaterally for 60 min; they were then fixed and imaged by confocal microscopy. Scatter plots show the degree of colocalization of IgG with Tf. The horizontal bars in the graphs indicate mean Mtarget protein. (e and f) Gene suppression of Rab25 (e) but not Rab11a (f) inhibits transcytosis of NIP-IgG (shaded bars) in both directions across the cell. NIP-IgG-IHH is used as control. Results are mean ± SD. (e, n = 6; f, n = 4). (g and h) Gene suppression of Rab11a inhibits NIP-IgG (shaded bars) recycling in the basolateral direction only. NIP-IgG-IHH (open bars) is used as control. Results are mean ± SD (n = 4). *, P < 0.001. (i and j) Doxycycline-induced silencing of Rab25 or Rab11a does not divert FcRn to the lysosomal compartment. Polarized cells treated or not treated with doxycycline were fixed and immunostained with LAMP1. The degree of colocalization of FcRn with LAMP1 was estimated using the Manders coefficient as described (see Materials and methods). Bar indicates mean Mtarget protein.
