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. 2017 Aug 25;45(18):10824–10836. doi: 10.1093/nar/gkx734

Figure 3.

Figure 3.

Tandem-affinity purification of Nob1p-containing pre-40S particles. Purification was performed using extracts from the TetO7-RIO1, NOB1-FPZ strain, grown in YPD (+ Rio1p) or in YPD plus doxycycline for 16 h (– Rio1p) to repress RIO1 expression. (A) Western analysis of tagged Nob1p purification. Aliquots from the initial extract (S10), flow-through of the first IgG-sepharose column (FT1), eluted sample from the IgG-sepharose affinity column (ELppx), flow-through of the second anti-Flag column (FT2), eluted samples from the anti-Flag column (E1–E5) were subjected to western analysis using anti-Flag antibodies. The positions of Nob1p-FPZ and Nob1p-Flag are indicated. (B) Northern analysis of 20S pre-rRNA, 18S and 25S rRNA levels in aliquots of the S10, ELppx and E3 samples from purification experiments performed with the TetO7-RIO1 strain grown in the presence of doxycycline as control (left), the TetO7-RIO1, NOB1-FPZ strain grown in the absence (middle) or presence (right) of doxycycline. Quantifications of the 20S/(18S+20S) and the (20S+18S)/25S ratios were performed by phosphorimager analysis. (C) Northern blot analysis of initiator tRNA levels in aliquots of the E3 anti-Flag affinity column elutions from the three purifications described in (B). Aliquots of S10 and polysomal gradient fraction (PolyS) are used as control.