Molecular imager fx pro plus system

To obtain nuclear protein extracts from MSCs, 24 h after culture on substrates with distinct stiffness, cells were washed with PBS and then scraped in Laemmli buffer. The subcellular fractionation was done following the protocol described in^{75 (link)}, and nuclear fractions were collected into microtubes and then heated at 95 °C for 5 min. Next, the samples were spun down and passed ten times through a 25 G needle and further analyzed by Western blot. Proteins were separated by SDS-PAGE [12.5% (w/v) acrylamide–bisacrylamide (Bio-Rad) gels] and transferred onto PVDF membranes that were subsequently probed with specific antibodies against H4K16ac (Abcam) and H4 (Cell Signaling) followed by the incubation with the respective alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch). The membranes were incubated for 5 minutes with enhanced chemifluorescence substrates (ECF – GE Healthcare) and imaged in a Molecular Imager FX Pro Plus system (BioRad) using the Quantity One software (BioRad). The acquired images were analyzed with Image Lab software, version 5.1 (BioRad).

Translocation analysis was performed by a combination of two previously described triplex oligonucleotide displacement assays [38 (link), 39 (link)] with some modifications. Plasmid pLKS5 carrying a triplex binding site 2093 bp downstream of the recognition sequence for enzyme EcoR124I and the triplex-forming-oligonucleotide TFO14 (5’ TTCTTTTCTTTCTTCTTTCTTT 3’) were used [39 (link)]. Covalently closed pLKS5 DNA and ³²P-labelled TFO14 were mixed in equimolar amounts (50 nM) in MM buffer (25 mM MES, pH 5.5, 12.5 mM MgCl₂) and incubated at 20°C overnight. The resulting triplex (10 nM) was pre-incubated with methylase (100 nM) and HsdR (250 nM) at 20°C in R buffer (50 mM Tris–HCl, pH 8.0, 10 mM MgCl₂, 1 mM DTT) for 5 min. Reaction was initiated by addition of ATP to a final concentration of 4 mM. The reaction was quenched after 1-h incubation at 20°C by addition of GSMB buffer (15% (w/v) glucose, 3% (w/v) SDS, 250 mM MOPS, pH 5.5, 0.4 mg/ml bromophenol blue) and the reaction products were analysed by electrophoresis in 1.5% (w/v) agarose gels [40 mM Tris–acetate, pH 5.5, 5.0 mM sodium acetate and 1.0 mM MgCl₂] at 10 V/cm for 3 h at 4°C. Wet gels were exposed on an imaging plate (BAS-IP MS 2025, Fuji film) and visualized in a Molecular Imager FX Pro Plus system (Bio Rad).