Figure 3. HEMA induces IB degradation and DNA binding of NF-B. (A) The cells were treated with 10 mM HEMA for the indicated periods of time and (B) with 0–10 mM HEMA for 180 min. Cytosolic extracts were analyzed by Western blotting with anti-IB antibodies. The lower panel shows a Western blot anti-tubulin as control for protein loading. Experiments were performed 3 times, and a representative result is shown. (C) EMSA from cells treated with 10 mM HEMA for the indicated period of time with or without PDTC. Total cell extracts were prepared and analyzed by EMSA with a ³²P-labeled oligonucleotide probe containing a NF-B-binding site. The middle portion of the autoradiograph shows the same cell extracts incubated with a 50-fold molar excess of unlabeled (cold) NF-B oligonucleotide.