FIG. 3.

Mak11-associated complexes do not contain late pre-60S factors. (A) Proteins purified in association with Mak11-TAP from wild-type (wt) cells or cells depleted for Rlp24 or Nog1 (14 h in glucose medium) were separated on a 5 to 20% polyacrylamide gradient gel and stained with colloidal Coomassie blue. Proteins identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, listed in Table 2, are indicated. (B) The presence or absence of proteins in TEV eluates from purifications using Mak11-TAP and Rlp24-TAP was determined by immunoblotting with specific antibodies. (C) The presence of Mak11 in complexes purified using Rlp24-TAP, Nog2-TAP, and Arx1-TAP was tested by immunoblotting, with Nog1 as a positive control. (D and E) RNAs associated with Mak11 complexes were enriched in the TEV protease eluate from a TAP-Mak11 (LMA326) purification. The recovered RNAs were extracted with phenol-chloroform and tested by primer extension (D) or Northern blotting (E) with the same oligonucleotides as those used for Fig. 1.