Pcdna3.1d v5 his

Screening constructs used for detection of cis-splicing and correct trans-splicing were designed and cloned according to the protocol of Bauer et al. [21 (link)]. Concisely said, the KRT14-scRTM carries a 5′ split-GFP part consisting of the first nucleotides (nt: 1–336) from a full length acGFP (pacGFP vector, Clontech, Mountain View, CA, USA) and a BD sequence that specifically targets Intron 7 of KRT14 cloned into the pcDNA3.1D/V5-HIS (Invitrogen, Carlsbad, CA, USA) backbone. The KRT14-scMG spanning exon 5 to intron 7 was generated using a KRT14 exon 5 forward primer (5′-GATCAAGCTTCACCACAGAGGAGCTGAACCGCGAGGTGGC-3′) and a KRT14 intron 7 reverse primer (5′-GATCGGATCCGGGGAAGAGGTGGGAAGAGGACGTTACC-3′) for amplification via GoTaq DNA polymerase (Promega, Madison, MI, USA). Afterwards, the amplified PCR product was cloned into the screening vector backbone downstream to the CMV-promoter (pcDNA3.1, Invitrogen, Carlsbad, CA, USA) using HindIII and BamHI restriction sites [21 (link),28 (link)]. The KRT14-scMG also carries the 3′ split-GFP portion consisting of the rearward part of acGFP (nt: 337–720) to enable a reconstitution of the full length acGFP upon accurate trans-splicing.

Total RNA was isolated and reverse transcription was performed followed by quantitative real-time PCR with SYBR Green qRCR mixture as described. PCR-based approaches were used to clone FBXL-2 into pcDNA3.1D/v5-his (Invitrogen) for constitutive expression in cells. All mutant constructs were generated using PCR-based methods with appropriate primers. RAW 264.7 cells were plated in 48 well plates at the density of 2.8 × 10⁵ cells per well, and the cells were cultured for 18 h. Then, 12 h after transfection, cells were moved to six-well plates, delivered into different groups, and incubated for further 18 h.