Phosphorylation of p38, ERK1/2, Akt/GSK3 and activation of NF-κB and CREB by rHagB stimulated DC is dependent on TLR4. DC from WT, TLR2−/−, TLR4−/−, MyD88−/− and TRIFLps2 mice were stimulated with 40 µg/ml rHagB for 10, 30, 60 or 120 min. Following stimulation, cells were lysed and whole cell lysates were assessed for (A) ERK1/2 and p38 and (B) Akt and GSK3α/β (C) p65 NF-κB (Ser 536), IκBα and CREB phosphorylation by Western blot. Total p38 (A) and GSK3β (B) were used as loading controls. Unstimulated DC (far left lanes) or DC stimulated with 100 ng/ml E. coli K12 LPS for 60 min (far right lanes) were used as controls. Results are representative of two independent experiments.