Fig 3. ZC3H11 co-purifies with the HSP70 3’ -UTR.

(A) Schematic representation of the reporters used for the HSP70 mRNP purification. The control reporter (right) lacks the AUU repeat that is bound by ZC3H11. (B) Northern blot showing the purification of both 3SBP-CAT mRNAs; details as in Fig 2. (C) Western blot showing specific co-purification of V5-ZC3H11 with 3SBP-CAT-HSP70 mRNA. The figures below the lanes show the relative loading in cell-equivalents. For the beads, about 10⁹ cell-equivalents were loaded. TR: trypanothione reductase (with an additional cross-reacting band); S9: ribosomal protein S9 (which detects two bands). A single V5-tagged band was observed. (D) Summary for four independent purifications. Results for the CAT baits are for four experiments each; those for TR and S9 are for all eight experiments. (E) The method cannot be used to identify proteins that bind to many other mRNAs in addition to the target mRNA. The same experimental set-up as in (B), but in cells expressing V5-MKT1. For the beads, about 10⁹ cell-equivalents were loaded.