Vibra cell processor

E. coli BL21 (DE3) cells carrying the plasmids of interest were grown in LB at 37 °C to an OD₆₀₀ of 0.6. Then, IPTG was added at a final concentration of 1 mM to induce protein expression, the temperature was decreased to 26 °C and the culture was incubated for 6 h. Samples (10 mL) were taken at 0 and 6 h of induction and centrifuged. The pellets were resuspended in 1 mL of Tris-HCl buffer (10 mM, pH 7.8) supplemented with protease inhibitor (NZYTech) and lysed by sonication on ice (35% amplitude, 3 s ON plus 9 s OFF for a total of 5 min ON) using a microtip probe linked to a Vibra-cell processor (Sonics). After sonication, samples were centrifuged and resuspended, and the protein of the soluble and insoluble fractions was quantified using the Bradford reagent (Panreac). The expression levels were evaluated through SDS-PAGE (4% stacking gel and 10% running gel). Soluble and insoluble protein fractions were mixed with 2 x sample buffer (65.8 mM Tris–HCl pH 6.8, 26.3% glycerol, 0.01% bromophenol blue, 2.1% SDS, 5% β-mercaptoethanol) and denaturated at 95 °C in the heating block for 5 min. The protein ladders used were NZYColour Protein Marker II (NZYTech) and Blue Prestained Protein Standard—Broad Range (NEB). The gel was stained with Coomassie Blue R-250 for 15 min and de-stained using distilled water.

Human dermal microvascular endothelial cells were cross-linked with 1% formaldehyde (Sigma-Aldrich, United Kingdom) for 10 min before being quenched with 0.125 M glycine for 5 min. Cells were then lysed in SDS lysis buffer containing 1% SDS, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris–HCL (pH 8.1) and 1× complete, mini, EDTA-free protease inhibitor cocktail (Roche, United States) for 10 min on ice. Cell lysates were sonicated into 500 bp fragments with a Vibra-Cell processor (Sonics, United States). Sheared chromatin was cleared of debris and incubated overnight at 4°C with either rabbit IgG (Sigma-Aldrich, United Kingdom), or 2 μg rabbit polyclonal anti-NF-κB p65 antibody (Santa Cruz, United States). After washing, the protein-DNA crosslinks were reversed and DNA was eluted in 240 μL elution buffer containing 1% SDS and 0.1 M sodium bicarbonate (NaHCO₃). DNA purification was performed using the QIAquick PCR purification kit (Qiagen, Germany) according to the manufacturer’s instructions. qRT-PCR was performed using the primers spanning two NF-κB binding sites near the LRG1 transcription start site (TSS). Binding sites located in fragments (−2000 to +2000 from TSS) that interact with LRG1 gene were predicted by motif analysis using FIMO (Grant et al., 2011 (link)).