Pcmv3tag

The gH (GP75), gL (GP115) and gO (GP74) ORFs were additionally separately cloned into expression vectors that also tagged the C-terminal domain for easy detection of the recombinant protein in transfected cells. For GFP tagged gH, the GP75 ORF (missing the stop codon) was PCR amplified as a BamH I fragment using primers FgHBm and RgHBmNostop (S1 Table) and cloned inframe into GFP fusion expression vector pAcGFP-N1 (Clontech) cut with BamH I. This introduced a GFP tag in-frame into the C-terminal domain of the gH ORF and placed the GP75 under a HCMV MIE promoter control. This modified plasmid was designated pAcGFPNgH. For mCherry tagged gL, the GP115 ORF (missing the stop codon) was PCR amplified as a Hind III fragment using primers FgLHd and RgLHdNostop (S1 Table) and cloned inframe into mCherry fusion expression vector pmCherry-N1 (Clontech) cut with Hind III. This introduced a mCherry tag in-frame into the C-terminal domain of the gL ORF and placed the GP115 under a HCMV MIE promoter control. This modified plasmid was designated pmCherryNgL. A GP128 FLAG tagged vector was generated by PCR using the primer set GP128F and GP128R (S1 Table) cloned as a BamH I fragment into pCMV3Tag (Agilent Technology). The GP128 ORF PCR lacked a stop codon and the BamH I site enabled inframe cloning that introduced a 3xFLAG tag into the C-terminus of GP128 (pFLAGGP128).

Expression vectors were constructed as follows: myc-tagged human TSHZ1, TSHZ2, TSHZ3 and mutated or deleted TSHZ2 from pCMV-3Tag (Agilent Technologies); DsRed-tagged human TSHZ2 from pDsRed-monomer (Clontech); GAL4-fused TSHZ2 from pBIND (Promega); HA-tagged or FLAG-EGFP-tagged human GLI1 and GLI2 and FLAG-tagged human CtBP2 from pCMVTNT (Promega); and EGFP-tagged GLI1 and GLI2 from pEGFP-C (Clontech). Mutated and deleted TSHZ2 constructs were generated via PCR-amplification. Luciferase reporter vectors were constructed as follows: pG5TKluc from UAS-containing pG5luc (Promega) modified by the introduction of the thymidine kinase minimal promoter (TKmini); wtGBSx4TKLuc and mutGBSx4TKLuc from the luciferase reporter pGL3 (Promega) modified by the introduction of four copies of the wild-type or mutated GLI-binding site of the human CXCR4 promoter. Lentiviral vectors expressing either human TSHZ2, GLI1 or LacZ were constructed from the CSII-CMV-MCS-IRES2-Bsd plasmid, which was kindly provided by Dr. Hiroyuki Miyoshi (RIKEN BioResource Center, Japan).