Fig. 3.

Butanol fraction recapitulates Nexrutine® in modulating NFκB signalingin PC-3 cells. A:Both Nexrutine® and butanol fraction treatment reduces protein levels of pAkt, p65 and pIκbα in PC-3 cells. Immunoblot analysis ofnuclear and cytoplasmic extracts prepared from PC-3 cells treated with Nexrutine® and fractions(2.5mg/ml for 24 hr)using pAkt, p65 and pIκBα. Equal amounts of the extracts were fractionatedona 10% SDS-polyacrylamide gel transferred onto a nitrocellulose membrane. The blotted membrane was blocked with 5% nonfat dry milk in TBS containing 1% Tween-20 (blocking solution) and incubated with indicated antibodies followed by incubation with HRP conjugated anti-rabbit IgG antibody (sigma) in blocking solution. Bound antibody was detected by enhanced chemiluminescence using Western lightning Western chemiluminescence reagent plus (enhancedluminol) following the manufacturer's directions (Perkin Elmer Lifeand Analytical Sciences, Shelton, CT). All the blots were stripped and reprobed withβ-actin to ensure equal loading of protein. Each experiment was repeated thrice using differentsets of extracts. The blot shownis a representative three different experiments. B: Nexrutine® prevents IκBα degradation. LNCa Pand PC-3 cells were transfected with pIκB EGFP vector and after 48 hr of transfection cells were stimulated with TNFα (20ng/ml for 30 min asapositive control for NFκB activation). One group of cells were preincubated with Nexrutine (2.5 μg/ml) prior to stimulation withT NFα and observed under fluorescence microscope. C: Inhibition of NFκB transcriptional activity by Nexrutine® and its fractions. Transient transfections were performed with NFκB reporter plasmid (1 μg/well) and pRLTK plasmid (20 ng/well) as described in the materials and methods using Lipofectamine reagent. Twenty-four hours after transfection, the cell swere treated with Nexrutine® and fractions(2.5 μg/ml) for 2hr. Luciferaseactivity was measured using the Dual-luciferase Reporter assay system (Promega Corporation Inc., Madison,WI) in triplicate samples containing equal amounts of protein. Renilla luciferase activity was used to normalize transfection efficiency. Results are expressed as the ratio of firefly luciferase to Renilla luciferase at equal amounts of protein. The data shown here are representative of three independent experiments conducted with two different preparations of plasmids. D: Inhibition of NFκB DNA binding by Nexrutine® and its fractions. EMSA of nuclear extracts prepared from PC-3 cells treated with Nexrutine® and its fractions (2.5 μg/ml for 2 hr). Five microgram extract was incubated with NFκB consensus oligonucleotide as radiolabeled probe and the DNA-protein complexes were resolved on a 4% nondenaturing gel by electrophoresis and subject to autoradiography. The blot shown here is representative of two independent experiments.[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]