Effect of MRB10130 depletion on TbRGG2 protein–protein interactions. (A) Schematic representation of the RESC complex divided into the GRBC and REMC subcomplexes (Ammerman et al. 2012, 2013; Aphasizheva et al. 2014; Read et al. 2016; McAdams et al. 2018). Solid lines (strong) or dotted lines (weak) indicate the strength of interaction observed in published yeast two-hybrid screens (Ammerman et al. 2012; McAdams et al. 2018). Thick lines represent interactions in both directions, whereas thin lines represent interactions that occurred in one direction of the screen. (B) PF T. brucei cells harboring both the MRB10130 RNAi construct and the TbRGG2-HTM construct were used to assess the proteins associated with the REMC subcomplex upon depletion of MRB10130. Cells were grown for 2 d in the absence or presence of tet, followed by IP of TbRGG2 using the myc tag. Bound protein was eluted from myc beads using TEV protease cleavage, and elutions were then analyzed using western blot with a subset of REMC and GRBC protein antibodies. Biological replicate experiments were performed and qRT-PCR was used to validate the level of MRB10130 knockdown (14%–21% remaining). (C) Quantification of western blots in B using BioRad Image Lab software. Protein levels were normalized to amount of TbRGG2 for that immunoprecipitation (IP). The normalized protein levels from the tet+ IP were then compared to that of the tet− IP (which was set to 100%) to calculate the protein associated with TbRGG2. Bar graphs represent the average and standard deviation of two biological replicates.
