FIG. 4.

Pam₃Cys induces NF-κB nuclear translocation and phosphorylation of IKK-α/β in mouse embryonic stem cell (mESC) line E14. Once cells reached confluence, 10 μg/mL Pam₃Cys or 1 μg/mL of lipopolysaccharide (LPS) was added. mESCs were harvested 30 min or 1 h later. Cytoplasmic (A and C) and nuclear (B and D) fractions were quantified, run on SDS-PAGE gel and blotted with anti-NF-κB p65, PARP, or total ERK1/2 antibodies. Autoradiographs were quantified by densitometry (C and D). Expression of PARP was used as a measure of cytoplasmic contamination of nuclear material. (E) Expression of MyD88. (F) IKK-α/β phosphorylation at 15 and 30 min with Pam3Cys and at 30 min with poly I:C. Anti-total IKK-α/β is shown. Anti-β-actin was used as a loading control. Results are representative of at least 3 experiments each.