The pEGFP-C1 vector (Clontech) comprises of an EGFP coding sequence under the control of a CMV promoter and a Kozak translation initiation site. Downstream of the EGFP sequence is the multiple cloning site containing unique BglII, SacI, HindIII and ApaI restriction sites. Contruction of pEGFP-C1/JV 59 GS/lacZ was as follows; The JV 59 GS sequence was amplified from the JV full length cDNA clone [7] using Bio-X-Act DNA polymerase (Bioline) with the primers 59 GS F (59-AACTGCA-GATCTTAATAAGTGAATGAAGACTTTGACGAT-39), containing the BglII restriction site (bold) and two in-frame translation termination codons (underlined) to ensure that translation of the EGFP sequence did not carry over to the 59 GS, and 59 GS R (59-AACTGCAAGCTTCTGCAGGACACAATGAGG-39), containing theHindIII restriction site. The JV 59 GS amplicon was ligated to the pEGFP-C1 vector, following restriction enzyme digestion of both amplicon and vector with BglII and HindIII restriction enzymes, and the ligated DNA used to transform E.coli Top10 (Invitrogen). This intermediate construct was named pEGFP-C1/ JV 59 GS. The lacZ coding sequence was amplified from the pSVb-Gal reporter vector (Promega) using Bio-X-Act DNA polymerase and the primers lacZ F (59-AACTGCAAGCTTGA-TATGGGGGATCCCGTCGTTTTACAACG-39), containing the HindIII restriction site (bold) and a kozak translation initiation site (underlined), and lacZ R (59-AACTGCGGGCCCTTAT-TATTTTTGACACCAGACCA-39) containing the ApaI restriction site (bold) and translation termination codons (underlined). The lacZ amplicon was ligated to the pEGFP-C1/JV 59 GS vector following restriction enzyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The construct was verified by sequencing. Construction of pEGFP-C1/IRES/lacZ was as follows; The EMCV IRES sequence was amplified from the pIRES2-EGFP vector (Clontech) using Bio-X-Act DNA polymerase and the primers IRES Bgl F (59-ACTCGAAGATCTTAA-TAGAGCTTCGAATTCTGCAGTCGA-39), containing the BglII restriction site (bold) and translation termination codons (underlined) to prevent carry over translation as before, and IRES Sac R (59-ACTCGAGAGCTCTGTGGCCATATTATCATC-GTG-39), containing the SacI restriction site (bold). The IRES amplicon was ligated to the pEGFP-C1 vector follwing restriction Whole cell lysate was collected at the following time points following CHX treatment: 0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr. Bradford analysis was performed on the lysates to ensure equal loading. Following Western analysis the ECL treated membrane was exposed to film for 1 min. Molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g008 enzyme digest of both amplicon and vector with BgIII and SacI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The lacZ amplicon described previously was ligated to the intermediate pEGFP-C1/IRES vector following restriction enzyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10.