FIGURE 3.

Homozygous deletion of Dhs is embryonically lethal in mice, whereas heterozygous Dhs knock-out animals are viable and show compensatory up-regulation of the hypusine system. A, representative litter size of the indicated breeding. Note that no Dhs^−/− pups were born after Dhs^+/− × Dhs^+/− breeding. B–D, mRNA levels of Dhs, Dohh, and eIF-5A1 in different organs of wild type or heterozygous mice as assessed by quantitative real time PCR. Expression values were plotted relative to the expression of the housekeeping gene 18S rRNA. E and F, expression of eIF-5A1 protein in different organs using Western blot. Protein expression was quantified relative to the loading control α-tubulin using the Odyssey Infrared Imaging System. G, two-dimensional Western blot detecting different eIF-5A1 forms in the outlined organs. H and I, analysis of MEFs isolated from Dhs-deficient Dhs^p/+ and Dhs^p/p-CAG-cre/Esr1⁺ mice as well as wild type Dhs^+/+; CAG-cre/Esr1⁺ animals after 4-OHT treatment in vitro. H, mRNA expression status of Dhs after 100 nm 4-OHT treatment at the indicated time points relative to the housekeeping gene 18S as assessed by quantitative real time PCR. I, two-dimensional Western blot for eIF-5A1 after treatment with 4-OHT (100 nm; 8 days) or incubation with 50 μm GC7 (2 days). Significances were calculated using the unpaired t test and marked with an asterisk if significant (***, p < 0.001; **, p < 0.01; *, p < 0.05). ⁺, wild type allele; ⁻, deleted allele. Colored arrows in the representative blots correspond to the different eIF-5A1 forms as outlined in the schematic plot on the left: black = fully hypusinated Lys⁵⁰, pH 5.2; red = unmodified Lys⁵⁰, pH 5.1; blue = unmodified Lys⁵⁰ plus acetylated Lys⁴⁷, pH 5.0. qPCR, quantitative PCR. n.d., not detectable.