Hrp coupled antibodies

Histone extracts were isolated from harvested cells using the EpiQuick Total Histone Extraction Kit (Epigentek) according to the manufacturer’s instructions. Nuclear extracts were isolated from harvested cells using standard nuclear fractionation protocol (Abcam). Protein concentrations were determined by the Lowry protein assay and 2 μg of histone or nuclear extract was used per well. Samples were run on 4–12% Bis-Tris gels (NuPAGE, Thermo-Fisher) in MES-SDS running buffer (NuPAGE, Thermo-Fisher). After 90 min of transfer at 30mV, nitrocellulose membranes were stained overnight at 4°C with anti-H3K27me3 (Cell Signaling, #9733; dilution 1/250), anti-H3 (Active Motif, #61475; dilution 1/5000), anti-Ezh2 (Cell Signaling, #5246; dilution 1/500) and anti-TBP (Cell Signaling, #44059; dilution 1/5000) diluted in blocking buffer (TBS, 0.1% Tween, 5% BSA). Protein detection was performed using HRP-coupled antibodies (Cell Signaling) and the ECL Prime detection reagent (GE Healthcare). Membranes were imaged and quantified using the G:BOX Chemi XT4 and associated software (Syngene).

For haematoxylin and eosin staining as well as immunohistochemistry, liver biopsies were
routinely fixed in formaldehyde and embedded in paraffin. Immunohistochemistry
and immunofluorescence microscopy was performed as previously described.25 (link),26 (link) For ultrastructural analysis, biopsies were fixed in
glutaraldehyde and embedded in epon or processed from paraffin blocks to epon.
Thin sections were analyzed in an electron microscope (JEM 1400, JEOL, Freising,
Germany).
Mouse monoclonal antibodies were against the lipid droplet-associated protein perilipin 2 (AP 125, Progen Heidelberg, Germany) and cytokeratin 7 (Clone OV-TL 12/30, Dako, Agilent Technologies, Santa Clara, 95051 CA, USA); additionally, rabbit antisera were used against SCYL1 (Atlas Antibodies N3C2, Biozol, Eching, Germany). Secondary anti-mouse and anti-rabbit hrp-coupled antibodies were from Cell Signaling, Danvers, MA 01923, USA.