Figure 3

RNA degradation is restricted to the lysis step prior to TAP purification. Strains RJY933 (Pbp2p-TAP), RJY358 (wt, untagged strain) and RJY929 (Nrp1p-TAP) were lysed in a French Press and processed up to the IgG immunopurification as indicated in Methods. Samples from supernatant of 20000 × g (S20) spin and pellet of 200000 × g (P200) spin, as well as input material (IgG input) and flow through (IgG FT) from the IgG immunopurification step were phenol extracted. 8 μg of the extracted RNA were loaded onto 1.2% agarose-formaldehyde gels and blotted onto nylon membranes. 8 μg of total RNA (P/C lysis RNA) from strain RJY933 (Pbp2p-TAP) prepared with a phenol extraction method [18] were loaded in parallel as control for intact RNAs. The positions of the 18S and 25S ribosomal RNAs in the methylene blue staining and the PDA1 mRNA hybridization signal are indicated.