FIG. 4.

Western blot detection of ORF73 protein with anti-Myc ascites and sera raised against ORF73 polypeptides. A eukaryotic expression vector (pA3M) containing ORF73 of both strains of HVS and KSHV was used to transiently transfect HEK293T cells. At 24 h after transfection, 10⁶ cells were harvested, lysed in SDS sample buffer, and resolved by SDS-8% PAGE. The resolved protein was transferred to a nitrocellulose membrane electronically, and the blot was used for the detection of ORF73 polypeptides. (A) Detection of proteins by anti-Myc monoclonal antibody. Lane Vector contained lysates from HEK293T cells transfected with empty vector pA3M. Lanes C488, A11, and LANA contained lysates from HEK293T cells transfected with expression constructs containing ORF73 of HVS C488, HVS A11, and KSHV, respectively. (B) The blot was stripped and reprobed with anti-C488 polyclonal serum from a squirrel monkey and showed cross-reactivity with C488 and A11 proteins. (C and D) Immunoblots showing cross-reactivity with anti-A11 and anti-LANA sera, respectively. Numbers at right indicate kilodaltons.