Kwikquant ultra digital ecl substrate solution

Subcellular fractions were isolated using Cell Fractionation kit (#9038S, Cell Signaling Technology) followed the manufacturer’s manual. 10 μg of nuclear fractions were loaded to each well of Bolt 4–12% Bis-Tris Plus Gels (# NW04125BOX, Thermo Fisher Scientifics) for SDS-PAGE and transferred onto 0.45 μm PVDF membranes (#IPVH15150, Millipore). As primary antibodies anti-Histone H3 rabbit polyclonal Ab (#07–690, Sigma-Aldrich, 1:2000), anti-Lamin B1 Rabbit mAb (HRP Conjugate) (#15068S, Cell Signaling Technology, 1:5000), anti-NF-κB p65 rabbit mAb (#8242S, Cell Signaling Technology, 1:1000), anti-Bsg rabbit mAb (#MA5–32534, Thermo Fisher Scientific, 1:1000) and anti-Hk2 rabbit mAb (#2867, Cell signaling Technology, 1:1000) were used, followed by goat anti-rabbit IgG HRP linked Ab (#7074S, Cell Signaling Technology, 1:2000). Blots were developed using the SuperSignal West Femto Maximum sensitivity Substrate Solution (#34095, Thermo Fisher Scientific) or KwikQuant Ultra Digital-ECL Substrate Solution (#R1002, Kindle Biosciences). CL-XPosure Film (#34090, Thermo Fisher scientific) or KiwiQuant® imager (#D1001, Kindle Biosciences) were used for signal detection. Data quantification was done using Fiji software (Schindelin et al., 2012 (link)).

Cells were lysed in 2-mercaptoethanol (BioRad) containing NuPAGE LDS sample buffer (Thermo Fisher Scientific). Equal amounts of each sample were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were then blocked with TBST containing 5% nonfat dried milk and incubated with primary antibodies overnight at 4°C. Following three washes in TBST, blots were incubated in horseradish peroxidase-conjugated secondary antibodies, then washed thoroughly before visualization using KwikQuant Ultra Digital-ECL Substrate Solution (Kindle Biosciences, LLC). Samples for Figure 6 were run contiguously on the same gel (no APV on one gel, and +APV in another gel). Phospho-specific antibody blots were stripped and reprobed for total CaMKII.