FIG 1.

ORF9b is ubiquitinated and degraded through the proteasomal pathway. (A) Proteasomal inhibitor MG132 increased the expression of ORF9b but not ORF3a. (B) Proteasomal inhibitors, but not other inhibitors, increased ORF9b expression. The ORF9b-HA-tag expression vector was transfected into HEK293T cells, and then the cells were treated with 10 μM MG132, Bortezomib, Carfilzomib, BafiloMycim AI, Vinblastine, or DMSO for 12 h prior to harvest. The cell lysates were analyzed using immunoblot (IB). (C) MG132 stabilized ORF9b expression in cells treated with CHX. ORF9b-HA was transfected into HEK293T cells for 24 h, and the cells were treated with or without 10 μM MG132 for 10 h, then 50 μg/mL cycloheximide (CHX) was added. The cells were harvested at different time points, and their lysates were analyzed. ORF9b (D) but not ORF3 (E) are ubiquitinated. HEK293T cells transfected with ORF9b-HA or ORF3-HA plus Ub-Flag or the empty vector were treated with 10 mM MG132 for 12 h prior to harvest. Co-IP (with anti-HA) and IB analysis were performed.