Figure 4.

Blockade of RAGE modulates activation of NF-κB after massive hepatectomy. (A) EMSA. Nuclear extracts were prepared from the remnants of the indicated mice and the EMSA was performed. The illustrated bands are representative of n = 4–6 mice per condition. **, P < 0.05 versus PBS treatment/85% resection. (B) Supershift assay. Remnants retrieved from mice undergoing 85% hepatectomy in the presence of sRAGE at 2 h were retrieved and subjected to incubation with anti-p50, anti-p65, or both anti-p50 and anti-p65 IgG before EMSA. (C) Immunohistochemistry. Hepatic remnants at 2 and 8 h were retrieved and subjected to immunohistochemistry with anti-p65 NF-κB subunit antibodies. Bar, 50 μm. (D) Proliferation. Hepatic remnants were retrieved and subjected to immunohistochemistry with anti-PCNA IgG and mean numbers of PCNA⁺ cells were determined from n = 10 fields per section/mouse. The indicated results are representative of n = 5 mice per condition. Bars, 80 μm. (inset) 160 μm. (E) Western blotting of hepatic remnant lysates was performed at the indicated times using anti-cyclin D1 IgG. The illustrated bands are representative of n = 3–4 mice per condition. (F) Apoptosis. Hepatic remnants were retrieved and subjected to TUNEL assay and mean numbers of TUNEL⁺ cells determined from n = 10 fields per section/mouse. The indicated results are representative of n = 5 mice per condition. Bar, 40 μm. (G) Western blotting on remnants was performed using anti-activated caspase-3 IgG. The illustrated bands are representative of n = 5 mice per condition. In all cases, bands were scanned into a densitometer and relative pixel units of band density reported. *, P < 0.05 versus PBS/85% resection.