Distamycin A disrupts binding of an inducible protein-DNA complex containing NF-κB to an AT-rich region of the P-selectin promoter.

Abstract

<p><b>A.</b> As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a>, lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or LPS/Dist A (Dist), then subjected to RNA extraction. The same blot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a> was hybridized with a radiolabeled probe for HMGA1 (and an 18S probe as loading control). This experiment was repeated two separate times. <b>B.</b> Nuclear extracts from BAEC cells (Lanes 1–7) and primary murine lung endothelial cells (MLEC, Lanes 8–12) (“Nuc Ext”) with (Lanes 3–7, 9–12) or without (Lanes 2,8) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and vehicle (V, lanes 3 and 10 (or in the presence of TNF-α without vehicle, Lane 9)) or increasing concentrations of Dist A (D₁ = 10 µM, D₂ = 20 µM, Lanes 4–5, 11–12) as well as with an identical competitor (IC, Lane 6) and a non-identical competitor (NIC, Lane 7). (* represents the inducible, specific complex seen following TNF-α treatment; “→” represents disruption of the TNF-α-inducible complex following addition of Dist A (Lanes 4–5 compared with Lane 3 and Lanes 11–12 compared with Lane 9–10). All of the binding studies were repeated at least two separate times. <b>C.</b> Nuclear extracts from BAEC cells (“Nuc Ext”) with (Lanes 3–7) or without (Lane 2) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and antibodies to the NF-κB family members p50 and p65 (lanes 4–5), or unrelated and control antibodies (Ets-1 and IgG control respectively, lanes 6–7). (* represents the inducible, specific complex seen following TNF-α treatment; “←” represents supershifted band/disruption of the TNF-α-inducible complex following addition of p50 and p65 antibodies (Lanes 4-5 compared with Lanes 6–7). All of the binding studies were repeated at least two separate times.</p