35s methionine cysteine mix

Mitochondrial translation products in control and patients fibroblasts were pulse-labelled for 1 h with 200 µCi/ml ³⁵S methionine/cysteine mix (Perkin Elmer) in Dulbecco’s Modified Eagle’s medium (Sigma) lacking methionine and cysteine. The media was supplemented with 100 µg/ml of emetine to inhibit cytosolic translation [essentially as described in Sasarman and Shoubridge (2012) (link)]. Protein extracts (15 µg) were electrophoretically separated by 15–20% gradient SDS-PAGE and visualized using a Typhoon FLA 9500 instrument.

The T7 promoter was appended 5′ of the mad1⁺ transcription start site by PCR. Precise sequences are available in Appendix Table S6. Full‐length mad1⁺ was amplified from cDNA generated using the SuperScript IV First Strand Synthesis System (ThermoFisher). Mad1 fragments 3′ of the intron were amplified from genomic DNA. PCR fragments were purified using the Wizard SV Gel and PCR Clean‐Up System (Promega). In vitro transcription was carried out with the HiScribe T7 ARCA mRNA Kit (with tailing) (New England Biolabs) using between 25 and 70 ng/μl template DNA. Reactions were run at 32°C or 37°C for 2 h. RNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs). RNAs were mixed and diluted as required before adding them to rabbit reticulocyte lysate (Promega). Translation reactions contained amino acid mix without Methionine, approx. 1 mCi/ml ³⁵S‐Methionine/Cysteine mix (Perkin Elmer, NEG772007MC), 0.2 U/μl SUPERase•In RNase Inhibitor (ThermoFisher), and between 0.35 and 40 ng/μl RNA. Incubation was at 30°C for 1 h 30 min.