Fig 6.

(A) No effect of 35B2 on steady-state levels of MCP in ORF40-transfected cells. 293T cells were transfected with empty vector (vec) (lane 1) or with phCMV-ORF40 (lanes 2 to 4), cultured in the presence of DMSO (lanes 1 and 2), 20 μM 35B2 (lane 3), or 20 μg/ml ACV (lane 4), and harvested at 44 h after transfection. MCP was detected by immunoblotting using rabbit anti-GST-ORF40 antibody and peroxidase-conjugated anti-rabbit IgG. (B) No effect of 35B2 on steady-state levels of scaffold protein in transfected cells. 293T cells were transfected with empty vector (lane 1) or with pCMV-ORF33.5F (lanes 2 and 3), cultured in the presence of DMSO (lanes 1 and 2) or 20 μM 35B2 (lane 3), and harvested at 44 h after transfection. Scaffold protein was detected by immunoblotting using anti-FLAG tag antibody and peroxidase-conjugated anti-mouse IgG. (C) No effect of 35B2 on the interaction between MCP and scaffold proteins in transfected cells. 293T cells were transfected with phCMV-ORF40 (lanes 1 and 2), with phCMV-ORF40 and pCMV-ORF33.5F (lanes 3 and 4), or with pCMV-ORF33.5F (lanes 5 and 6), cultured in the presence of DMSO (lanes 1, 3, and 5) or 35B2 (lanes 2, 4, and 6) and harvested at 44 h after transfection. The cell extracts were reacted with anti-GST-ORF40 antibody and protein G. Samples after immunoprecipitation (IP) were analyzed by immunoblotting (IB) using the anti-FLAG antibody. (D) No effect of 35B2 on steady-state levels of MCP in infected cells. HEL cells infected with V-Oka were harvested at 22 h after infection. MCP was detected as described for panel A.