The incubation was terminated by 10-fold dilution with ice-cold PBS buffer before the cells were washed and submitted to FACS analysis.
The supernatant was aspirated and the cell layer was washed twice with ice-cold KRH buffer before the cells were treated for 30 min with 0.5 ml 0.05 N NaOH in phosphate-buffered saline.
After 48 hours, the cells were washed briefly in phosphate buffer before extracting the cells with 0.5% Triton X-100 in cytoskeletal buffer (2 mM MgCl2, 10 mM EGTA, 60 mM Pipes pH 7.0) for 5 min at 37°C, as described previously [18].
The injection phase was 120 seconds followed by 60 seconds of buffer flow before the cells were washed at high flow rate.
For the caspase-3 assay, 1 × 10 Vero cells were resuspended in cold cell lysis buffer before the reaction mix was added.
The cells were washed using Olink buffer B and stained with DAPI (1 μg/mL) for 1 minute before washing with 0.01% buffer B. The cells were dried in the dark and cover-slipped using Vectashield HardSet Mounting Medium.
The protease inhibitors aprotinin, pepstatin, leupeptin, as well as the reducing agent dithiothreitol were added to this buffer before cell lysis.
That half-children, half-adult buffer before the news".
For immunoprecipitation experiments, Protein A beads (GE Healthcare) were incubated with 3 µg anti-Dnmt2 antibodies and 1 µg/ml insulin (Sigma) for 1 hour at 4°C, washed 3×5 minutes in IP-buffer before whole cell protein extracts were added to the beads for 3 hours at 4°C.
For SQR activity, 40 μg of protein lysate extracted before the assay (Cell Lysis Buffer, Cell Signaling) were added to 1 ml of the SQR assay buffer (10 mM KH2PO4, pH 7.8, 2 mM EDTA, 1 mg/ml BSA, 80 μM DCPIP, 4 μM rotenone, 0.2 mM ATP and 10 mM succinate) and incubated at 30 °C for 10 min.
