1. TbERP1, TbERP2, TbERP3, and TbERP8 are codependent for stability.
TbERP1, TbERP2, TbERP3, and TbERP8 are codependent for stability. The in situ TbERP epitope-tagging constructs were transfected into each of the TbERP RNAi cell lines. Specific dsRNA was induced for 72 h, and whole-cell lysates were prepared from control (Tet-) and induced (Tet+) cells, as indicated in the column labels. Lysates were fractionated by SDS-PAGE (1 × 107 cell equivalents per lane). Membranes were first immunoblotted with anti-TY or anti-HA to evaluate TbERP protein stability under RNAi, as indicated in the row labels. Blots were then stripped and reprobed for cytosolic Hsp70 as a loading control. Two biological replicates are presented (total n = 3). qRT-PCR analyses confirmed that RNAi knockdown is specific, and mRNA levels of opposing tagged TbERP constructs were unaffected, even under significant protein depletion (not shown).
Emilia K. Kruzel et al. mSphere 2017; doi: /mSphere