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. 2013 Feb 14;62(3):811–824. doi: 10.2337/db11-1652

FIG. 2.

FIG. 2.

PA28 KO mice show impaired proteasome function and accumulation of polyubiquitinated proteins in the liver. A: Mice were genotyped by PCR analysis as previously described (18,19). Genomic DNA extracted from mouse tails was analyzed by PCR. Primers used for genotyping were 5'-TTTCCTGTACGTGACTTCCATCCTGTTG-3' (primer 1), 5'-GGTCCACATACAATAAAGACATGGGCTG-3' (primer 2), and 5'-GATTGTGGTCCTCCTGCAACGCCTAAA-3' (primer 3). Primers 1 and 3 amplified 1.2-kb fragments from the wild-type PA28α allele. Primers 1 and 2 amplified 2.2-kb fragments from the mutant allele. Primers used for genotyping were 5'-CCGGGACAATAAGACACATCACTC-3' (primer 1), 5'-TTGTCCCTCCCTCCAGTTGTCTAA-3' (primer 2), and 5'-GATCCCCTCAGAAGAACTCGTCAA-3' (primer 3). Primers 2 and 3 amplified 0.9-kb fragments from the wild-type PA28γ allele. Primers 1 and 3 amplified 1.9-kb fragments from the mutant allele. B: RT-PCR analysis of total RNA prepared from liver of PA28 KO mice (n = 7). N.D., not determined. Transcripts of mouse PA28α, -β, and -γ were examined by quantitative RT-PCR. Data were normalized according to GAPDH mRNA level and presented as a value relative to that for WT mice. C: Western blot analysis of extracts prepared from the liver of PA28 KO mice. The blot was probed with the anti-PA28α, anti-PA28β, and anti-PA28γ antibodies. D: Body weights of WT and PA28 KO mice fed an STD or the HFD. ○, WT mice fed the STD (n = 5); ●, WT mice fed the HFD (n = 4); □, PA28 KO mice fed the STD (n = 4); ■, PA28 KO mice fed the HFD (n = 4). Data represent means ± SE. E: Comparison of liver proteasome activity in WT (n = 13, STD; n = 4, HFD) and PA28 KO (n = 11, STD; n = 4, HFD) mice. Data represent means ± SE. *P < 0.05. F: Proteasome activity was evaluated in the muscles isolated from 32-week-old WT mice (n = 5) and PA28 KO mice (n = 4). The activity was normalized to the total protein content. Data represent means ± SE. *P < 0.05. G: Western blot analyses of total ubiquitinated proteins in the livers of WT and KO mice. Livers were isolated from 32-week-old WT and PA28 KO mice fed the STD or the HFD for 28 weeks (from 4 to 32 weeks of age). Quantitation of ubiquitinated proteins levels is normalized to GAPDH and is represented as means ± SE (n = 3 per group). *P < 0.05.