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. 2018 Dec 6;100(3):721–736. doi: 10.1093/biolre/ioy225

Figure 3.

Figure 3.

Expression of PP1γ1 in Rescue IV. (A) Design of PP1γ1 Rescue IV construct mimicking PP1γ2 mRNA. The transgene bears exons 1–7 of PP1γ1 and exon 8 of PP1γ2 with 5΄ and 3΄ UTRs. The arrows indicate the mutations GT to GC and CAG to ATT. The region between the arrows is the extended exon 7 sequence specific to PP1γ1 mRNA. It is driven by the Pgk2 promoter for testis-specific expression. (B) Results of RT-qPCR showing comparable levels of PP1γ mRNA in testes of Rescue IV “Ppp1cc (–/–), Pp1γ1” in comparison to the heterozygous control “Ppp1cc (+/–)” and the PP1γ2 rescue “Ppp1cc (–/–), Pp1γ2”. Error bars indicate the standard error of the mean SEM (n = 3). (C) Quantification of PP1 levels expressed per 10 μg of testis extract of control and rescue mice was obtained by intensity analysis of immunoreactive bands of western blot. Panel on the right shows the western blot analysis of Rescue IV testis. Ten micrograms of testis extracts from Ppp1cc (+/–), Ppp1cc (–/–), Pp1γ2, and Rescue IV “Ppp1cc (–/–), Pp1γ1” along with 10 and 5 ng of His-PP1γ1 and His-PP1γ2 were analyzed by western blot and probed with anti-PP1γ1 and anti-PP1γ2 antibodies. The blots were re-probed with antibodies against β-Actin to confirm equal protein loading. The blots are representative of three different experiments. (D) Quantification of PP1 levels expressed per 2 × 106 sperm of control and rescue mice was obtained by intensity analysis of immunoreactive bands of western blot. Panel on the right shows the western blot analysis of Rescue IV sperm extracts. A total of 2 × 106 sperm from Ppp1cc (+/–), Ppp1cc (–/–), Pp1γ2, and Rescue IV “Ppp1cc (–/–), Pp1γ1” along with 5 ng of His-PP1γ1 and His-PP1γ2 were analyzed by western blot and probed with anti-PP1γ1 and anti-PP1γ2 antibodies. The blots were re-probed with antibodies against β-Tubulin to confirm equal protein loading. The blots are representative of three different experiments. (E) Phoshatase activity in sperm. Protein phosphatase activity in the soluble fraction (supernatant) and insoluble fraction (pellet) of sperm from Ppp1cc (+/–), Ppp1cc (–/–), Pp1γ2, and Ppp1cc (–/–), Pp1γ1 rescue mice expressed in millimoles/minute/extract. Total phosphatase activity represented as black bars is the combined phosphatase activity of PP1γ and PP2A. Activity of PP1γ was measured by deducting the activity remaining after incubation with recombinant I2 from the total activity. The phosphatase activity values are mean ± SEM obtained from animals aged 3–5 months. Ppp1cc (+/–) (n = 6), Ppp1cc (–/–), Pp1γ2 (n = 2), Ppp1cc (–/–), Pp1γ1 (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 and n.s indicates not significant.