FIG 5.

Culture supernatants of M2L-deleted vaccinia virus-infected CEFs do not interfere with the B7–CTLA4/CD28/PD-L1 interactions. (A) Culture supernatants collected from uninfected CEFs, as negative controls, or from CEFs infected with either MVA or a double-deleted (TK⁻ RR⁻, VV-DD-Luc) or a triple-deleted (TK⁻ RR⁻M2L⁻, VV-TD-Luc) Copenhagen vaccinia virus were concentrated and treated as described in the legend of Fig. 3 The blot was incubated with 2.5 μg/ml of CD80-Fc, and the binding of the fusion protein was monitored using an HRP-conjugated anti-Fc antibody and an enhanced chemiluminescence. kit. (B) The supernatants (but unconcentrated) used for the experiment described in panel A were tested in the same competition assays as described in the legend of Fig. 4. The optical density values recorded were normalized using the mean of the triplicate values of uninfected CEFs as 100% of the signal. Each represented value is the mean (± standard deviation) of three normalized measurements.