Fig. 3. Domains in TLK1 and TLK2 molecules required for in vivo phosphorylation and subcellular localization. Chimeric proteins constructed by exchanging the N- or the C-termini between TLK1 and TLK2 or their kinase-dead mutants were expressed as HA-tagged proteins in T. brucei. Cells were boiled in SDS sampling solution and immunoblotted with mAb against HA for the slower-migrating band as evidence for in vivo phosphorylation. The cells were also fixed, immunostained with mAb against HA and counterstained with DAPI for localizing the HA-tagged chimeric proteins in cells (also see supplementary material Fig. S4).