Fig 1: RCC1L isoforms and mitochondrial ribosomal subunits in isokinetic sucrose gradients.Mitochondrial ribosome profile in parental and RCC1L overexpressing cells after induction with 10ng/ml doxycycline for 3 days. Equal amounts of mitochondrial lysates from each of the four cell lines were separated on a 10–30% (v:v) isokinetic sucrose gradient and fractions were analysed by immunoblotting. In the immunoblots of endogenous RCC1L, the 50 kDa band (black arrowhead) contains isoforms RCC1LV1 and RCC1LV2, while the 37 kDa band (red arrowhead) corresponds to isoform RCC1LV3. The STREP2-FLAG-tagged RCC1L proteins (RCC1LV1, RCC1LV2 and RCC1LV3) are also marked (grey arrowheads) in each case. Antibodies against structural components (uL11, bL12, ICT1) and assembly factors (MALSU1) of the mtLSU along with GTPases (MTG1, MTG2, GTPBP10) and components of the pseudouridylation module (TRUB2, RPUSD3 and RPUSD4) involved in mtLSU biogenesis were used for immunodetection of proteins of interest. In the case of mtSSU analysis, immunoblot analysis was performed using antibodies against structural components (uS17, mS18b, mS29) and related GTPases (ERAL1, C4ORF14). Immunoblots for additional mtLSU and mtSSU proteins are shown in S4 Fig. Transparent blue, orange and yellow colours mark the fractions where the 28S mtSSU (fractions 8–9), 39S mtLSU (fractions 6–7) and 55S monosome (fractions 4–5) peak, respectively whereas non-assembled subunit peaks are left unmarked (fractions 10–14). Equal volume of each fraction was loaded for all cell lines.