Effect of MRB10130 depletion on MRB3010 and MRB11870 protein–protein interactions. PF T. brucei cells harboring both the MRB10130 RNAi construct and the MRB3010-PTP (A,B) or MRB11870-PTP (C,D) construct were used to assess the proteins associated with the GRBC subcomplex in the absence of MRB10130. Cells were grown for 2 d in the absence or presence of tet, followed by IP of PTP-tagged protein using IgG. Bound protein was eluted from IgG beads using TEV protease cleavage, and elutions were 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 (30%–45% remaining). (B,D) Quantification of western blots in (A,C) using BioRad Image Lab software. Protein levels were normalized to amount of PTP-tagged protein 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 either MRB3010 or MRB11870. Bar graphs represent the average and standard deviation of two biological replicates.
