FIGURE 5.

AKIP1 mediates NF‐κB regulation via the Akt/IKKβ pathway during the EMT. A, Serum‐starved HeLa cells were pretreated with PI3K inhibitor Ly294002 (40 μM) or IKK inhibitor Bay117082 (15 μM) for 4 hours, followed by stimulation with AKIP1 protein (80 ng/mL) for 4 hours. Cell lysates were harvested and the levels of indicated proteins were measured using western blot analysis. B, pGL4.32 (luc2NF‐κB‐RE/Hygro) vector and pRL4‐TK Renilla luciferase constructs were used for co‐transfection of HeLa cells. The cells were further transfected 24 hours later with empty vector (EV) or AKIP1 expression plasmid. Some of the AKIP1‐transfected cells were pretreated with Ly294002 (40 μM) or Bay117082 (15 μM) for 4 hours. The dual luciferase reporter assay was performed 24 hours following transfection of AKIP1. EV‐transfected cells treated with TNFα (40 ng/mL) for 1 hour after transfection were used as the positive control. ** indicates P < .01. C, The HeLa cells were transfected with AKIP1 siRNA to knock down AKIP1. After 72 hours transfection, AKIP1 protein (80 ng/mL) was used to stimulate HeLa cells for 4 hours and the cell lysates were analysed using western blot