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. 2019 Oct 10;10:1278. doi: 10.3389/fphys.2019.01278

FIGURE 4.

FIGURE 4

Decreased PI31 levels but normal proteasome activity in Fbxo7LacZ/LacZ testis. (A) Immunoblot analysis for various proteins in whole testes lysates from WT, heterozygous, and homozygous LacZ mice at 8.1 weeks of age, as indicated. NS = non-specific band in Fbxo7 (Santa Cruz Biotechnology, sc-271763) Western blot. (B) Quantitation of Fbxo7 expression from immunoblots, relative to GADPH loading control; gray bars; n = 10 WT, 8 heterozygous, 7 homozygous LacZ mice. RT-qPCR of Fbxo7 relative to three housekeeping genes (cyclophilin, GAPDH, and actin); black bars; n = 4 for each genotype). Images were analyzed in ImageJ. ∗∗∗p < 0.001 One-way ANOVA with Dunnett’s multiple comparisons test. (C) Quantitation of PI31 expression from immunoblots, relative to GADPH loading control; gray bars; n = 10 WT, 8 heterozygous, 7 homozygous LacZ mice. RT-qPCR of Psmf1/PI31 relative to three housekeeping genes (cyclophilin, GAPDH, and actin); black bars; n = 4 for each genotype). Images were analyzed in ImageJ. ∗∗p < 0.01 One-way ANOVA with Dunnett’s multiple comparisons test. (D) Immunoblot analysis for Fbxo7 (Laman Lab polyclonal antibody) and PI31 protein in whole testes lysates from mice harvested at the indicated days post-partum (p). (E) Immunoblot analysis for Fbxo7 (Santa Cruz Biotechnology, sc-271763) and PI31 protein in whole testes lysates from mice harvested at 19 and 24 days post-partum. Quantification of PI31 protein levels relative to WT GADPH levels for each sample is indicated. (F) Proteasome activity measured in whole testis extract for each genotype. Treatment with MG132 abolished the signal, confirming the specificity of the assay. n = 3 WT, 2 heterozygous, and 4 homozygous LacZ mice, each performed in triplicate ∗∗∗p < 0.0001.