Figure 7: Hausp point mutation strategy in knock-in mouse strain and its identification through characterization of hypoxia response in fibroblasts.

(a) The point mutation strategy used to generate Hausp K444R point mutant in mouse ES cells. (b) Identification of Hausp K444R mutation (A to G) in mouse skin fibroblast cells by sequencing and PCR analysis. cDNA products from wild-type (Hausp^+/+) and from K444R homozygous (HAUSP^K444R/K444R) were amplified by PCR using HAUSP typing primers to generate the 594 bp (WT) and 694 bp (K444R) products. (c) Real-time PCR analysis to examine the response of three mouse Hif-1α target gene expression under hypoxia. WT, wild type; Mut, point mutant; N, normoxia; H, hypoxia. Data from three independent experiments are expressed as mean±s.d. The asterisk (*) indicates statistical significance (P<0.05) between experimental and control cells, t-test. (d) Western blot analysis showed that the expressions of Hif-1α downstream targets were decreased in Hausp K444R mutant mouse skin fibroblasts under hypoxia. (e) Western blot analysis showed that the K48 polyubiquitination levels of Hif-1α were increased in the Hausp homozygous K444R mouse skin fibroblasts treated with MG132 under hypoxia compared to those in the wild-type Hausp mouse skin fibroblasts.