Mrps35

Specific proteins were detected using rabbit polyclonal antibodies against: MRPL44 (16394-1-AP), MRPL23 (11706-1-AP), TACO1 (21147-1-AP), MRPS35 (16457-1-AP), MRPS16 (16735-1-AP) (Proteintech, diluted 1:1,000), MRPS34 (Sigma HPA042112, diluted 1:1,000), and mouse monoclonal antibodies against: β-actin (8226), porin (14734), NDUFA9 (14713), complex II (14715), complex III (14745), COXI (14705), COXII (198286), COXIV (14744) and complex V subunit a (14748; Abcam, diluted 1:1,000), in Odyssey Blocking Buffer (Li-Cor). IR Dye 800CW Goat Anti-Rabbit IgG or IRDye 680LT Goat Anti-Mouse IgG (Li-Cor) secondary antibodies were used and the immunoblots were visualized using an Odyssey Infrared Imaging System (Li-Cor). Examples of uncropped blots are shown in the Supplementary Fig. 8. Tissue-specific analysis was performed using a Proteintech mouse tissue blot (Cat. No M10005).

Heart and liver mitochondria, 1.2 mg, were lysed in the presence of 1% n-Dodecyl β-D-maltoside (Sigma). Lysates were loaded on 10–30% sucrose gradients and separated by centrifugation overnight as previously described [17 (link),38 (link)]. Gradient fractions were collected as 750 μl aliquots. RNA was extracted from one third of each fraction by using the TRIzol LS Reagent (Invitrogen) according to the manufacturer’s recommendations. The samples were subsequently treated with DNase I and used for cDNA synthesis. The transcript abundance in each fraction was assessed by qRT-PCR analysis using the Taqman probes listed in S1 Table. The remaining two thirds of each fraction (500 μl) were precipitated with trichloracetic acid, resolved by SDS-PAGE and ribosome-containing fractions were detected by immunoblotting using antibodies specific for individual proteins from the 28S (MRPS35, Proteintech) and 39S (MRPL37, Sigma) ribosomal subunits.