Immunoblot analyses of mitochondrial proteins isolated from TbTim9 RNAi, TbTim10 RNAi, and TbTim8/13 RNAi cells were performed using antibodies for TbTim17, which is a polytopic MIM protein. (G to I) TbTim8/13 are displayed here twice: once with the inner cysteines of the twin CX3C motif WZ811 (C2 and C3, Rabbit Polyclonal to TAS2R49 left model) and once with the outer cysteines of the twin WZ811 CX3C motif (C1 and C4, right model). The models are presented in the same order from left to right as shown in Fig.?1E to ?toG.G. Download FIG?S1, PDF file, 1.1 MB. Copyright ? 2018 Smith et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S2? Effect of small TbTim overexpression on cell growth. Cell growth analysis of parasites that express (A) TbTim9-Myc, (B) TbTim10-Myc, or (C) TbTim8/13-Myc in the absence (uninduced) or presence (induced) of doxycycline (1.0?g/ml) for 6?days. The log of the cumulative cell numbers was plotted versus time (in days). Download FIG?S2, PDF file, 1.2 MB. Copyright ? 2018 Smith et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3? Quantitation of immunoblots for mitochondrial proteins after osmotic swelling and proteinase K treatment. The signal intensities of Myc, Cyt cells and from the cells expressing TbTim10-Myc. Bound proteins were analyzed on an SDS-PAGE gel and stained with Coomassie blue. Lanes for individual samples were excised from the gel, proteins were digested by trypsin, and peptides were analyzed by mass spectrometry. A complete list of the identified proteins is presented in Table?S3. Download TABLE?S2, PDF file, 1.1 MB. Copyright ? 2018 Smith et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S3? Complete list of the identified proteins. Download TABLE?S3, XLSX file, 0.02 MB. Copyright ? 2018 Smith et al. This content is WZ811 distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S4? Expression of the fusion proteins TbTim9-Gal4BD, TbTim10-Gal4BD, and TbTim8/13-Gal4BD in yeast. (A to D) Yeast cell cotransformed with the vacant pGADT7 and pGDKT7 plasmid constructs were grown in synthetic defined (SD) Ctrp/Cleu medium or Ctrp/Cleu/Chis medium containing 0 to 5?mM AT to show that yeast cells cotransformed with vacant plasmids are unable to grow in triple-knockout medium. (E) A strain (Y2H Gold) transformed with the TbTim9, TbTim10, and TbTim8/13 fusion constructs in the pGDKT7 plasmid was produced in synthetic defined (SD) medium lacking tryptophan (Ctrp). The pGDKT7 plasmid adds a Myc epitope to Gal4BD. Cells (optical density [OD] of 3) were pelleted by centrifugation, resuspended in 200?l of 0.1?M NaOH, and incubated at room temperature for 5?min. Cells were recovered by centrifugation and lysed with reducing Laemmli buffer (300?l), and soluble proteins were subjected to immunoblot analysis using WZ811 anti-myc antibody. A 20-l volume of soluble proteins was loaded per lane. A Y2H strain that was not transfected with any plasmid was produced in yeast extract-peptone-dextrose-adenine (YPDA) medium, processed similarly, and used as a control. A nonspecific band present in all lanes is usually indicated by an asterisk (*). Download FIG?S4, PDF file, 0.1 MB. Copyright ? 2018 Smith et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S5? Wild-type and uninduced RNAi control results were comparable. (A) Scatterplot representation of quantitative reverse transcriptase PCR (qRT-PCR) analysis of TbTim9, TbTim10, and TbTim8/13 mRNA levels in wild-type, TbTim9 RNAi, TbTim10 RNAi, and TbTim8/13 RNAi cells after 96?h either in the absence (uninduced RNAi) or the presence (induced RNAi) of doxycycline (1.0?g/ml). The mRNA levels of the target transcript in wild-type parasites were considered to represent 100%. Small TbTim transcript levels were normalized with the levels of the tubulin mRNA in each sample. Values were calculated from three impartial replicates (****, 0.0001), and error bars indicate standard deviations. (B) Mitochondrial protein analyzed.