Chemdoc imager

Binding between nucleosomes and isolated HAT module was performed by incubating 0–4 μM HAT with 50 nM nucleosome at 30 °C for 30 min in binding buffer (20 mM HEPES pH 7.5, 150 mM potassium Acetate, 4 mM MgCl₂, 0.2 mg/mL bovine serum albumin (BSA), 1 mM Dithiothreitol (DTT). The samples were loaded onto a 4 % TBE gel that had been pre-run for 60 min at 4 °C in 0.5 X TBE and then run for 90 min following sample loading. Complexes were visualized using SYBR Gold, which stains DNA. Experiments were done in triplicate and gel images were recorded with a ChemDoc imager (BioRad) and analyzed using Image Lab 6.1 by quantitating the unbound nucleosome band.

Protein, 20–30 µg, was separated by electrophoresis on 10–12% SDS-polyacrylamide gels at 60–100 mV and then transferred onto polyvinylidene fluoride (PVDF) membranes for 90 min on constant 200 mA at 4 °C. After protein transfer, membranes were blocked in 5% BSA + Tris-buffered saline plus Tween-20 (TBST) blocking buffer for an hour, then incubated with the respective primary antibodies overnight at 4 °C. Following 3 washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega,#W401B, Madison, WI, USA) or HRP-conjugated anti-mouse IgG secondary antibodies (BioRad, #170-6516, Mississauga, ON, Canada) (1:5000) for 1 h at room temperature. Signals were detected using ECL plus kit reagents (Perkin Elmer, Guelph, ON, Canada) on a Chem Doc imager (BioRad, Mississauga, ON, Canada). Primary antibodies used were: Anti-Caspase-12 (Abcam, #ab62484, Toronto, ON, Canada); Anti-KDEL (Abcam, #ab176333, Toronto, ON, Canada), which recognizes GRP94, GRP78, and PDI; Anti-XBP1(Abcam, #ab37152, Toronto, ON, Canada); Anti-ATF6 (Proteintech, #24169-1-AP, Rosemont, IL, USA); Anti-PERK (Proteintech, #20582-1-AP, Rosemont, IL, USA); Anti-IRE1α (Proteintech, #27582-1-AP, Rosemont, IL, USA).