FIGURE 6.

Time course of anserine production in lysates of control as well as UPF0586 protein-overexpressing COS-7 and HEK-293T cells. COS-7 and HEK-293T cells were transfected for 48 h with an empty vector (Control) or a vector encoding yeast, chicken, rat, or human UPF0586 protein (yUPF0586, chUPF0586, rUPF0586, or hUPF0586, respectively). Cell-free lysates (40 μg of protein) were incubated at 37 °C for the indicated times in the presence of 1 μm [¹H + ³H]SAM (100 pmol, ∼400 × 10³ cpm) and 20 mm carnosine. The formation of radiolabeled anserine was determined after its chromatographic separation from [³H]SAM. The presence of recombinant protein in tested lysates was verified by Western blot analysis, which was performed using 40 μg of total protein and an antibody against the His₆ tag. The figure shows results of representative experiments.