Figure 8. Effect of cytoplasmic TRAF4 on ERK signaling pathway.

(A): 40% and 100% confluent HeLa cells that never form TJs (Figure 6 D) were transfected with the Flag-TRAF4 expression vector (2.5 µg) or an empty vector (2.5 µg). Twenty-four hours after transfection, proteins were extracted, and analyzed by immunoblotting with antibody for phospho-ERK1/2. Total amounts of ERK1/2 were evaluated by reprobing the blots with pan-ERK antibody. TRAF4 expression was tested using 2H1 anti-TRAF4 antibody. Activation of ERK1/2 is dependent on cell confluency. (B): a similar experiment in non-confluent cell condition using increasing amounts of the Flag-TRAF4 expression vector (1 µg, 5 µg or 10 µg). Activation of ERK1/2 is dependent on the amounts of transfected Flag-TRAF4. (A and B): a representative western blot from four independent experiments is shown. Thus, TRAF4 favors ERK1/2 MAPK activation in proliferating HeLa cells.