Journal: Hepatology Communications
Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury
Figure Lengend Snippet: HFD feeding increased the translocation to the nucleus of p65 and expression of NFκB‐related genes in Irf3 −/− mice. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expressions of phospho‐IKKα∕β, IKKβ, phospho‐p65, p65, and IκBα were assessed by western blot and normalized to GAPDH, n = 4 per genotype. (B) Formalin‐fixed paraffin‐embedded sections of liver were deparaffinized, and localization of phospho‐p65 was assessed by immunohistochemistry. Images were acquired at 40×. White arrows indicate nonparenchymal cells, and black arrows indicate hepatocytes. Images are representative of triplicate images from eight mice per treatment group. Values represent means ± SEM. (C) Nuclear fractions were isolated from livers, and equal amounts of protein were assessed by western blot and probed against phosphor‐Ser 536 p65‐NFκB. Lamin A/C was used as a nuclei marker, and GAPDH was used as a cytosolic marker. (D) Expressions of CCL5, CXCL10, CXCL2, and CCL3 were analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values with different superscripts are significantly different from each other, P
Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).
Techniques: Translocation Assay, Expressing, Mouse Assay, Western Blot, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Isolation, Marker, Quantitative RT-PCR