Anti h3k4me3

Hippocampal tissue samples were thawed on ice then treated with 1% formaldehyde for five minutes and sonicated with the truChIPTM Tissue Prep Kit for SDS Chromatin Shearing (Covaris) and the Covaris® S2 Sonicator (Woburn, MA, USA) according to the manufacturer’s protocol. The EpiQuik™ Tissue Chromatin Immunoprecipitation Kit (Epigentek) was used to perform ChIP. After sonication, samples were divided and immunoprecipitated with ChIP-grade polyclonal antibodies anti-H3K4me3 (Epigentek cat # A-4033) and anti-H3K27me3 (Millipore cat #07–499). Two microarray experiments were performed, one for each methylation state using the same chromatin sample from the same mice for each. Immunoprecipitated samples were sent to ArrayStar Inc. (Rockville, MD, USA). ArrayStar performed whole-genome amplification, target preparation DNA labelling and array hybridization.

Molecular events in the kidneys of LPS-treated mice were monitored using a chromatin immunoprecipitation (ChIP) assay to immunopurify soluble chromatinized H3K4 me3 sequences using specific antibodies and the EpiQuik ChIP kit (EPIGENTEK, Brooklyn, NY, USA). The antibodies used included normal mouse IgG as the negative control, anti-RNA polymerase II as the positive control, and anti-H3K4 me3 (EPIGENTEK, Brooklyn, NY, USA). The DNA concentration was quantified using a NanoDrop UV spectrometer (NanoDrop Technologies, Wilmington, DE, USA). A SYBR green assay (LightCycler 480 SYBR Green I Master; Roche, Mannheim, Germany) was performed using the 7300HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using primers for mouse cathepsin L (EpiTect ChIP qPCR Assays, QIAGEN).