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. 2015 Dec 30;10(12):e0145783. doi: 10.1371/journal.pone.0145783

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© 2015 An et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 5 — (A) The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. (B) Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. (C) Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3L^cta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. (D) Codon optimization of M3L^cta to M3L^ttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3L^ttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. (E) Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTEN^flox/flox cells. Constructs were nucleofected into PTEN ^loxp/loxp mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. (F) The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, PTEN ^loxp/loxp cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (t _Shld1) and/or at different PTEN-concentrations (d _Shld1).