FIG. 8.
Effect of ubiquitin mutants on gB ubiquitination. To detect ubiquitin conjugates, 293T cells were transfected with expression vectors encoding the following HA-tagged forms of ubiquitin (HA-Ub): the wt, K48R mutant, or K63R mutant. Twenty-four hours later, the cells were infected with HSV-1 (10 PFU/cell) (+) or mock infected (−). Twenty-four hours postinfection, gB was immunoprecipitated from cell lysates. Western blotting (WB) was performed with the indicated antibodies (α-HA, MAb to HA; α-HSV-1, PAb to major HSV-1 glycoproteins). Circles identify the ubiquitinated forms of gB; values to the right of the circles indicate their apparent Mrs. Arrows point to the 170- and 160-kDa forms of ubiquitinated gB absent from the K63R sample. The values to the right identify the migration positions of molecular mass markers (in kilodaltons).