Bp2564 1

The assay was carried out as previously described (Wilson et al, 2011). 1 × 10⁶ cells were plated in a 10‐cm tissue culture dishes and cultured for 48 h. The adherent cells were trypsinized and re‐suspended gently in ice‐cold PBS. The cells were centrifuged at 300 × g, 4°C for 5 min, and the cell pellets were vortexed vigorously in 500 μl ice‐cold 60% methanol for 1 min. The suspension was then heated at 95°C for 3 min and sonicated. The extracts were centrifuged at 16,000 × g for 5 min at 4°C to remove cell debris, precipitated protein, and DNA. The supernatant was evaporated using centrifugal vacuum at 70°C and reconstituted in 25 μl nuclease‐free water.
Reaction mixtures contained primer, probe, and template at an equimolar final concentration of 0.4 mM. Standard dNTPs (Fisher Scientific, BP2564‐1) were included for the standard curve. DreamTaq DNA Polymerase (Thermo Scientific, EP0703) was added at 1 U/reaction to a final volume of 25 μl. Thermal profiling and fluorescence detection were performed using the Quant Studio 3 Real‐Time PCR System (Thermo Scientific). Raw fluorescence spectra for 6‐FAM were measured every 3 min.

RNA was extracted from INS-1 832/13 cells with TRIzol (Invitrogen #15596026) and 500 ng RNA was used for first-strand cDNA synthesis in a reaction containing 50 ng of random primers (Promega #C1181), 500 μM dNTPs (Fisher Bioreagents #BP2564-1), 1× first-strand buffer, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega #M1705).