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Effect of IGF-1 on p65 nuclear localization and NF-κB pathway activation in Panc02 cells.

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posted on 07.05.2014, 03:38 by Alison E. Harvey, Laura M. Lashinger, Drew Hays, Lauren M. Harrison, Kimberly Lewis, Susan M. Fischer, Stephen D. Hursting

A, Representative fluorescent images of p65 localization at various time points following IGF-1 treatment (400 ng/mL) using 300 nM DAPI alone, a p65 antibody alone (p65), or p65 antibody counterstained with DAPI (merged). Data representative of 4 separate assays. B, ELISA of p65 DNA binding in response to 4-hour IGF-1 treatment (400 ng/mL). C, NF-κB luciferase reporter assay after 6-hour IGF-1 treatment (400 ng/mL). *denotes significant differences between SF and IGF-1. D, PCR analysis of mRNA expression of NF-κB downstream gene targets after 18-hour IGF-1 treatment. E, ELISA measurement of RANTES, LIF, and VEGF (n = 3) expression in Panc02 supernatant after 24 hours of IGF-1 treatment. *denotes significant differences between SF and IGF-1 with respect to each respective gene or protein of interest. All experiments used 400 ng/mL of IGF-1. Bar graphs represent mean ± SEM (n = 3 separate experiments performed in triplicate for B, C, and D). SF, serum-free; IGF-1, insulin-like growth factor-1; DAPI, 4′-6-diamidino-2-phenylindole; RANTES, Regulated on Activation, Normal T cell Expressed and Secreted; LIF, leukemia inhibitory factor; VEGF, vascular endothelial growth factor.