reported relative to the replication of untreated cells, which was set at 100%. The viability of the replicon cells was measured using the WST-8 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19770275 cell counting kit according to the manufacturer’s instructions. Flow Cytometry to Detect Surface DHCR24 Cell surface expression of DHCR24 protein was assessed by binding studies with 2-152a MAb. In the negative control experiments, normal mouse IgG was used. For flow cytometry analysis, 1 106 cells were incubated with 1 g/mL 2-152a MAb, 152a Chimera Ab, or 152a scFv-hIgG-Fc at 4C for 2 h. The cells were then washed 3 times with PBS and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody or an Alexa Fluor 3 / 17 Surface DHCR24 Is a Target for HCV-Related HCC Therapy 488-conjugated goat anti-human IgG antibody at 4C for 1 h. The cells were washed again and analyzed using a FACSCalibur flow cytometer. When acquiring data on the flow cytometer, forward scatter versus side scatter plots were created and it was ensured that all the expected cell populations are visible by adjusting the individual FSC and SSC photomultiplier tube settings. Most of the debris, air bubbles and laser noise, were removed from analysis by setting and adjusting the FSC threshold. Next, the region around the major cell populations was inserted on the FSC vs SSC plot. The FL1 histogram data was made from the R1 cell population. Data were analyzed using CellQuest software, and the mean fluorescence intensity calculated. Black shades indicate the unstained cell population, the blue line indicate the isotype-reacted cell population and the red line indicate the cell population stained with 2-152a MAb or derivatives. Complement-dependent Cytotoxicity Assay HeLa, HepG2, and HuH-7 cells were incubated with 1 or 10 g/mL 2-152a MAb or normal IgG in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768759 the presence of guinea pig complement for 30 min at 30C. After incubation, cell viability was measured using the WST-8 cell counting kit according to the manufacturer’s instructions. Construction of a Chimeric Antibody containing the 2-152a MAb Antigen-binding Domain and the Human IgG Constant Domain Total RNA was extracted from hybridoma clone #2-152a, and then cDNAs of the variable regions of the light and heavy chains were obtained by RT-PCR. The VH and VL cDNAs were inserted into pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk, respectively. These recombinant vectors were co-transfected into HEK293 cells, and then the chimeric antibody was secreted into the culture medium as a soluble protein. The chimeric antibody was purified from the culture medium with protein A/G/L sepharose. Construction of a scFv Derived from 2-152a MAb and a Fusion Protein with Human IgG1-Fc Digitoxin web Single chain Fv fragments derived from 2-152a MAb were constructed by SOE-PCR and then inserted into pFUSE-hIgG1e4-Fc2. These recombinant vectors were transfected into HEK293 cells, and then scFv-human IgG1 Fc fusion proteins were secreted as soluble proteins into the culture medium. The chimeric antibody was purified from the culture medium using protein A/G/L sepharose. Western Blot Analysis Cells were lysed with 100 L of lysis buffer, and then 50 g of total protein was electrophoresed on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The blot was probed with 2-152a MAb to detect DHCR24 protein and with an anti-actin monoclonal Ab to detect actin as an internal 
loading control. 4 / 17 Surface DHCR24 Is a Target for HCV-Related HCC Therapy Construction of a DHCR24-expressing
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