FIGURE 2.

B3A reduces luciferase protein. a, TMP-B3A reduces luciferase signal of both wild type luciferase and luciferase-eDHFR fusion protein. The graph represents 5 independent replicates of cells treated with either DMSO or 200 μm TMP-B3A. All samples were measured with Luciferase Assay System (Promega) on a luminometer and normalized with a Bradford assay. b, B3A-containing ligands decrease luciferase signal. HEK-293T cells expressing HA-luciferase were treated for 4 h with 100 μm of the listed compounds. The bars represent the average and standard deviation of 4 independent replicates. All samples were measured as in a. c, Cbz-B3A does not inhibit luciferase enzymatic activity. Recombinant luciferase was incubated with compound for 10 min, then substrate was added and activity was measured. The bars represent the average and standard deviation of 5 independent replicates. d, Cbz-B3A reduces luciferase protein. HEK-293T cells expressing HA-luciferase were treated with 100 μm Cbz-B3A for 4 h. Cells were lysed and analyzed by immunoblotting with an anti-HA antibody. Both blots are from the same experiment and the same exposure. e, the graph represents quantification of d. f, Cbz-B3A does not reduce firefly luciferase mRNA. HEK-293T cells expressing HA-luciferase were treated with 100 μm Cbz-B3A for 4 h. Firefly luciferase mRNA was measured by quantitative RT-PCR and normalized to actin. The bars represent the average and standard deviation of 3 independent replicates. g, Cbz-B3A reduces Renilla luciferase. HEK-293T cells transfected with pRSV-Renilla were treated with 100 μm Cbz-B3A for 4 h. All samples were lysed in reporter lysis buffer and measured with substrate on a luminometer and normalized with a Bradford assay. The bars represent the average and standard deviation of 4 independent replicates. Significance was determined in comparison to DMSO; n.s., not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.