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. 2016 Sep 18;62(6):607–614. doi: 10.1262/jrd.2016-116

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©2016 Society for Reproduction and Development

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Fig. 2. — Generation of transgenic mice expressing polyadenylation-defective PAPOLB^D114A. (A) Immunoblot analysis of proteins synthesized in vitro. N-terminally FLAG- and HA-tagged PAPOLB (WT) and PAPOLB^D114A (D114A) were synthesized in rabbit reticulocyte lysates and were analyzed by immunoblotting using anti-HA antibody. ACTB was used as a control. (B) Polyadenylation assay. The polyadenylation activity of wild-type PAPOLB (closed circle) and PAPOLB^D114A (closed square) was monitored by measuring the incorporation of AMP from [α-³²P]ATP into an oligo(A)₁₂ primer at the incubation times indicated. Empty vector (open circle) was used as a control. (C) Physical map of the transgenic construct. The protein-coding regions of FLAG and HA tags and PAPOLB^D114A are indicated by open and filled boxes, respectively. The restriction enzymes are denoted as follows: A, ApaI; B, BamHI; E, EcoRI; H, HindIII; S, SalI; N, NotI. (D) Immunoblot analysis. Testicular protein extracts of wild-type (WT) and two transgenic mouse lines (Tg16 and Tg66) were probed with anti-HA and anti-PAPOLB antibodies. ACTB served as a control.