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Fig. 6. NF-B activity and keratin mRNA expression after ex vivo treatment of isolated acini with caerulein +/–MG-132. (A) Lanes 1-6: freshly isolated mouse pancreatic acini were treated with caerulein for 1-4 hours (37°C) followed by nuclear protein extraction and electrophoretic mobility shift assay (EMSA) as described in Materials and Methods. Binding in the presence of excess unlabeled NF-B-binding oligonucleotide (+probe, lane 6) is included as a specificity control. Lanes 7-12: acini were incubated in the presence or absence of caerulein for 2 hours (±MG-132 for 1 hour before the caerulein incubation) followed by nuclear protein extraction and EMSA as in lanes 1-6. (B) Pancreatic acini were incubated with caerulein (0.1 µM, 4 hours) with or without pretreatment with MG-132 (50 µM, 1 hour). Total RNA was isolated followed by real-time RT-PCR analysis of K8, K18 and K19 mRNA as described in Materials and Methods. Expression of each keratin mRNA was normalized to expression of the L7 gene (not shown). Results represent the mean±s.d. of three independent experiments. ^*P<0.01 vs. control; ^**P<0.01 vs. caerulein.

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