Figure 4. Vps35 overexpression does not restore pIgA transcytosis if PI3K is inhibited in MDCK-pIgR^Δ670–707 cells.

MDCK cells expressing pIgR^Δ670–707 were grown as a polarized monolayer on Transwells and infected with adenovirus carrying the myc-hVps35 gene together with an adenovirus carrying the tTA under a tetracycline repressible system (co-infection was required because these cells do not stably express tTA). A ligand transcytosis assay using ¹²⁵I-pIgA was performed on cells treated (+ LY) or untreated (− LY) with 50 μM LY294002. a, LY294002 treatment in cells overexpressing ~ 5-fold Vps35 (adenoviral-induced) abolished apical transcytosis restored by Vps35 overexpression. In the presence of the inhibitor, uninduced control cells (repressed with the antibiotic) showed no change on transcytosis, which remained at background levels. b, PI3K inhibition substantially increased basolateral recycling in uninduced cells and to a lesser extent in Vps35 overexpressing cells. c, PI3K inhibition reduced ligand degradation only in uninduced cells. The curves for uninduced cells are represented by a solid line and circles are used as symbols. For Vps35 overexpressing cells, the curves are dashed lines and diamonds are used as symbols. LY294002 treatment is denoted by filled symbols vs. empty symbols in untreated cells. Values are the mean ± SD (n = 3).