Characterization of hepatic zonation in mice by mass-spectrometric and antibody-based proteomics approaches.

Periportal and perivenous hepatocytes show zonal heterogeneity in metabolism and signaling. Here, hepatic zonation in mouse liver was analyzed by non-targeted mass spectrometry (MS) and by the antibody-based DigiWest technique, yielding a comprehensive overview of protein expression in periportal and perivenous hepatocytes. Targeted immunoaffinity-based proteomics were used to substantiate findings related to drug metabolism.

Activation of transcription factor circuity in 2i-induced ground state pluripotency is independent of repressive global epigenetic landscapes.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3β (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles.

Array-based Western-blotting reveals spatial differences in hepatic signaling and metabolism following CAR activation.

The complex three-dimensional architecture of the liver with its metabolically zonated lobules is a prerequisite to perform functions of metabolic conversion of endogenous and foreign substrates. The enzymatic competencies of hepatocytes differ between zones and dynamically adapt upon xenobiotic activation of the nuclear constitutive androstane receptor (CAR). Using the antibody-based DigiWest proteomics approach, the abundance and phosphorylation status of hepatocyte proteins isolated by laser capture microdissection from the periportal and pericentral regions of murine liver lobules were analyzed.

A high-throughput pipeline for validation of antibodies.

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS).

Antitumor effects of regorafenib and sorafenib in preclinical models of hepatocellular carcinoma.

The purpose of this study was to investigate the antitumor activity of regorafenib and sorafenib in preclinical models of HCC and to assess their mechanism of action by associated changes in protein expression in a HCC-PDX mouse model. Both drugs were administered orally once daily at 10 mg/kg (regorafenib) or 30 mg/kg (sorafenib), which recapitulate the human exposure at the maximally tolerated dose in mice. In a H129 hepatoma model, survival times differed significantly between regorafenib versus vehicle (p=0.

A bead-based western for high-throughput cellular signal transduction analyses.

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales.