FIG 12.

Silencing of PI3K and MEK inhibits the coimmunoprecipitation of phosphorylated PI3K and ERK signaling molecules with the V-ATPase pump. (A and B) LLC-PK cells were transfected with scrambled siRNA or siRNA against PI3K p85α (A) or MEK (B) and then incubated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA for the indicated times. Subsequently, the cell lysates were immunoprecipitated using antibodies specific for V₁ subunit E of V-ATPase, pPI3K, pAkt, and pERK. The coimmunoprecipitated products were analyzed by Western blotting to detect pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and V₁ subunit E by use of the relevant antibodies. GAPDH was used as a loading control.