Figure 1
Construction of short-lived Nef expression vector. Schematic representations of Nef-WT and Nef-CP used in this study (A). HEK293 cells were transfected with pNefmac239-WT or pNefmac239-CP. After a 48 h cultivation, HEK293 cells transfected with pNefmac239-CP were treated with 20 μM MG132 for 0 or 6 h. The cells were collected and subjected to SDS-PAGE and western immunoblot analysis to detect Nefmac239-WT and Nefmac239-CP using the anti-V5 antibody. β-Actin was also detected using the anti-β-actin antibody, as described in Materials and Methods (B). The relative band intensities of Nefmac239-CP and β-actin were quantified using Fujifilm Image Gauge software. The expression levels of Nefmac239-CP relative to those of β-actin treated with and without MG132 were calculated using the obtained intensities and compared (C).