Expression vectors for stable overexpression of the proteins Clover-PPARγ, roGFP2, and Grx1-roGFP2 were generated by replacing EGFP of pHR′SIN-cPPT-SE [22 (link)] by the appropriate coding sequences due to In-Fusion® (Clontech, Takara, Japan) recombination as previously described [23 (link)]. In brief, the plasmids pcDNA3-Clover [24 (link)], pDsRed-Monomer-C1-hPPARγ1 [25 (link)], and pQE-60_Grx1-roGFP2 [26 (link)] were used as templates. N- and C-terminally HA-tagged hPPARγ was generated by the same approach, elongated by an IRES sequence derived from the pLVX-TRE3G-mCherry (Clontech), and an additional Clover-sequence to generate a hPPARγ-IRES-Clover expression construct. Therefore, hPPARγ was fused to the HA-coding sequence 5′-ATGTACCCATACGATGTTCCAGATTACGCT-3′ at the N-terminus and to 5′-CCCCCTCCGCCCCCACCTTACCCATACGATGTTCCAGATTACGCTTGA-3′ at the C-terminus. Cysteine to alanine mutants of hPPARγ were created by mutagenesis of the TGX into GCX codons using PfuII polymerase (Agilent Technologies Deutschland GmbH, Böblingen, Germany). The same mutagenesis approach was used for the generation of serine to alanine (TCT to GCT) and serine to glutamic acid (TCT to GAG) mutants of hPPARγ.
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