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. 2019 Nov 26;20(23):5941. doi: 10.3390/ijms20235941

Figure 2.

Figure 2

Puf6 and Loc1 are important for the protein stability of Rpl43. (A) The protein levels of Rpl43A-HA (PKL349) and Rpl43B-HA (PKL350) in wildtype (BY4741), puf6Δ (KLY67), and loc1Δ (KLY218) were detected by Western blotting. The signals were quantitated by Image J. HA signals were normalized with Rpl8, and the relative ratios to WT were calculated. (B) 2μ PUF6 (PKL333) and LOC1 (PKL746) were transformed to wild-type cells containing RPL43B-HA (PKL563). The protein levels were checked by Western blotting. The relative ratios of Rpl43 in PUF6- or LOC1- overexpression strains to the vector-only control were calculated. (C,D) Cycloheximide was added to the cells and incubated for different times. Cell lysates were spun at 80,000 rpm for 60 min at 4 °C. The pellets (ribosome fraction) were re-dissolved in 1× Laemmli buffer. The proteins in the supernatant (free form) were precipitated by TCA and re-dissolved in 1× Laemmli buffer. The proteins were detected by anti-tubulin and anti-HA antibodies. The signals were quantitated by Image J.