Odyssey clx machine

This assay performed as described previously (Connelly et al., 2020 (link))(Piccolo et al., 2019 (link)). PCR-generated, biotin end-labeled 147 bp DNA probes (2 μg) were coupled to M280-streptavidin Dynabeads according to the manufacturer’s instructions (Invitrogen). All cytosines in the probes were either non-methylated or methylated and only occurred in a single sequence context (CG, CAC or CAT), sequences in the Key resources table. Bead-DNA complexes were then co-incubated with 20 μg of rat brain nuclear protein extract (Mellén et al., 2017 (link)) for 1.5 hours at 4◦C. Following extensive washing, bead-bound proteins were eluted using Laemmli buffer (Sigma) and resolved on a 4%–15% SDS-polyacrylamide gel (NEB). The presence of MeCP2 was assayed by western blot using MeCP2 (Sigma M6818; RRID:AB_262075) diluted 1:1000; with secondary detection employing Li-COR IRDye 800CW Donkey anti-Mouse (926-32212) diluted 1:10,000, then scanned using a LI-COR Odyssey CLx machine.

Cells and mammospheres were lysed in ice in a RIPA buffer (1% NP-40, 150 mM NaCl, 1% sodium deoxycholate, 0.1 % SDS, 25 mM Tris-HCl pH 7.6). The lysates were centrifuged and the cytoplasmic extract was collected in the resulting supernatant. Samples (20 μg/10 μL) were prepared from the cells and mammospheres. After electrophoresis on a 12% SDS-PAGE gel, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated in an Odyssey blocking buffer at room temperature for 1 h and then incubated overnight with primary antibodies. The antibodies were p65, LF-MA30327; GAPDH, LF-PA0018; Oct4, LF-MA30482 (AbFrontier, Seoul, Korea); LaminB, sc-365962; Nanog, sc-293121; Sox2, sc-365923 (Santa Cruz Biotechnology, Dallas, TX, USA); and c-Myc, 551101 (BD, San Jose, CA, USA). After the membranes were washed, they were incubated with IRDye 680RD and 800W secondary antibodies at room temperature for 1 h, and the signals were determined with an Odyssey CLx machine (Li-Cor, Lincoln, NE, USA).

IRDye 680RD DBCO (0.5 mg) (LI-COR, 429 nmol) was resuspended in 42.9 µL phosphate-buffered saline (PBS) for a concentration of 10 mM. The L5 linkers (Azide-DNA-RNA oligonucleotides) were ordered from IDT (Integrated DNA Technologies) and resuspended in PBS. Oligonucleotides were run through a Zymo RNA-clean-and-concentrator kit (purification was required for labelling), using ~7 µg oligonucleotide per column, and eluting at ~0.5 mg/mL (~40 µM) in water. Before binding to columns, we added ethanol to a final concentration of 67% instead of the 50% recommended by the manufacturer. During column purifications, washes were performed using an 85% ethanol in water solution made fresh each time, in place of the kit’s wash buffer. Five microliters of 10 mM dye (~50 nmol) was added to 10–150 µg purified oligonucleotide (~1–12 nmol) in PBS for a total volume of 200 µL and reacted for 2 h at 37 °C. Oligonucleotides were then run again through a Zymo clean-up kit and eluted in water. Concentrations were determined by A₂₆₀ ratio using an approximate ε = 368,050 M⁻¹. Oligonucleotides were diluted to 10 nM in ligation buffer (50 mM Tris pH 7.5, 10 mM MgCl₂, 16.7% PEG400), 1 µL was blotted onto a nylon membrane, and fluorescence was measured in an Odyssey CLx machine (LI-COR). This was typically ~15,000 fluorescence units/fmol for full labelling.

For immunoblotting, samples were separated on 10% or 4–20% gradient Novex^TM WedgeWell^TM Tris-Glycine gels (Thermo Fisher Scientific) for 40 min at 225V and transferred at 40V overnight at 4°C onto nitrocellulose membranes. Gels were stained with Coomassie following each transfer to verify normalization. The membranes were blocked in 5% non-fat milk (NFM) dissolved into 0.1% tween 20/PBS (PBST) at room temperature for 30 min. The membranes were incubated in primary antibodies diluted in 5% NFM/PBST at room temperature for 1 h or at 4°C overnight (see concentrations above), then washed 3x with PBST for 5 min each. The membranes were incubated with secondary antibodies diluted in 5% NFM/PBST at room temperature for 1 h (see concentrations above), washed 3x with PBST and 1x with PBS for 5 min each, then scanned with a LI-COR Odyssey CLx machine. Protein lysates were normalized by either method: staining gel with Coomassie before running the western blots (densitometry conducted with Adobe Photoshop), or blotting for pan-actin and normalizing the bands of interest to the pan-actin band intensity (densitometry conducted with Image Studio version 5.2).

Twelve microliters of supernatant or cell lysate were separated on a 4–12% Mini-Protean TGX gel (Bio-Rad) at 100 V for 1 h. Proteins were transferred onto a 0.2 μm nitrocellulose membranes (Li-Cor Biotechnology) at 100 V for 1 h. The membranes were blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h followed by incubation in PBS containing 5% BSA, 0.2% Tween-20, and either 1:1,000 rabbit polyclonal anti-Caspase-1 p20 (47 (link)), anti-IL-1β (70 (link)), anti-HMGB1 (abcam; ab18256), anti-PKR (phospho T446) (abcam; ab32036), anti-PKR (abcam; ab226819), anti-beta Actin antibody (abcam; ab8229), Anti-GFP antibody (abcam; ab6556), or 1:500 anti-Asc (Tyr-144) phospho-specific antibody (ECM Bioscience; AP5631) with rotation overnight at 4°C. The following day, donkey anti-rabbit IRDye 800CW and donkey anti-goat IRDye 680RD secondary antibodies (Li-Cor Biotechnology) were applied at a dilution of 1:15,000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx machine with auto exposure and high-quality setting.