Fig 2. Repression of the gene for the 40S r-protein eS4/S4 causes accumulation of extra-ribosomal uL18/L5, but not extra-ribosomal uL5/L11, uL4/L4, or uS4/S9.

P_gal-eL43/L43, P_gal-eS4/S4, and the parent strain BY4741 were grown in galactose medium and switched to glucose medium for the indicated time. Whole-cell lysates were fractionated on sucrose gradients and the indicated fractions of the gradients were analyzed by western blots probed with antisera for r-proteins uL18/L5, uL4/L4, eS4/S4, and uL5/L11 as indicated at each blot. (A) P_gal-eL43/L43 after 6 hours in glucose medium. All fractions from the top of the gradient through the polysome region are shown. (B) P_gal-eS4/S4 after 8 hours in glucose medium. The panel shows sections cropped from a western blot loaded with aliquots of each fraction from the top of the gradient through the polysome region. The blot was first probed with anti-uL18/L5, then with a mixture of antisera for uS4/S9 and uL4/L4 without stripping. Finally, the bottom of the blot was probed with anti-uL5/L11. (C) The parent strain (BY4741) after 0 and 16 hours in glucose. Aliquots of consecutive fractions from the top of the gradient and the 60S-80S region were analyzed by western blot. The top panel shows the blot probed with a mixture of antisera for uL4/L4 and uS4/S9. The middle panel shows the same blot after it was probed further with anti-uL18/L5 without stripping. The bottom panel shows the same blot after it was probed further with anti-uL5/L11. Uncropped images are shown in S1 Fig. The bands marked with a blue star in some panels are not related to uL18/L5 (see Fig 1 in Material and Methods). CE: cell extract.