eIF5A depletion does not affect the half-life of Tim50∆Pro protein nor the expression of the Tim50 and Tim50∆Pro copies. (A) Wild-type and tif51A-1 strains harboring Tim50-GFP (up) or Tim50∆Pro-GFP (down) were cultured in SGal medium until reaching post-diauxic phase at 25°C and transferred to 37°C for 4 h. Then cultures were treated with CHX (100 µg/ml) for 5 h and Tim50 protein levels were determined by western blotting using a GFP antibody. G6pdh levels were used as loading control. A representative image is shown from three independent experiments. (B) Tim50∆Pro steady-state protein levels were quantified from A. (C) Wild-type and tif51A-1 harboring Tim50∆Pro and an extra copy of wild-type Tim50 were cultured as in A. mRNA relative levels from wild-type TIM50 (left) and total TIM50 (wild-type TIM50 plus TIM50ΔPro, right) were determined by RT-qPCR. Data information: In B and C, results are presented as mean ± SD from three independent experiments. The statistical significance was measured by using a two-tailed paired Student’s t test relative to wild-type strain. n.s means no significant differences. Source data are available for this figure: SourceData FS5.