Fig. 4.
Subcellular localization and life cycle-dependent expression of TbPRMT6. (A) PF cells were separated into cytoplasmic and nuclear fractions. Mitochondrial extracts were also prepared in a separate fractionation scheme. Equal amounts of protein (20 μg) from whole-cell lysates (WCL), cytoplasm, mitochondria, and nuclei were fractionated on SDS-PAGE gel. Antibodies against Hsp70 (cytoplasmic), TbRGG2 (mitochondrial), and the RNA polymerase II CTD (nuclear) were used to monitor fractionation efficiency. TbPRMT1 and TbPRMT7 are two cytoplasmic PRMTs that were also used as controls. (B) Determination of TbPRMT6 levels in the procyclic form (PF) and bloodstream form (BF) life cycle stages of T. brucei. Total cell extract (equivalent of 5 × 106 cells) from 29-13 PF cells and single-marker BF cells were resolved by SDS-PAGE and immunoblotted using an antibody against TbPRMT6. As a loading control, levels of MRP2 (a protein known to be equivalent between PF and BF life cycles [92]) were also analyzed by Western blotting.