Figure 3. PFKFB3 S-glutathionylation induced by oxidative stress inside cancer cells.

(a) ROS levels in HeLa cells after treatment with the varying amounts of H₂O₂ and BCNU. Error bars denote SEM. (b) Detection of ROS-induced S-glutathionylation of cellular PFKFB3. PFKFB3-overexpressing HeLa cells and 293T cells were treated with or without 1.5mM Bio-GEE and 100 μM H₂O₂. Then, biotin-GSS-protein conjugates were precipitated using streptavidin-agarose. Immunoblotting shows PFKFB3 levels before (input, bottom) and after precipitation (upper) (60 μg of total protein per lane). (c) Detection of ROS-induced S-glutathionylation of endogenous PFKFB3 in HeLa cells. With HeLa cells transfected with the empty vector, an experiment similar to (b) was performed. (d) Detection of ROS-induced S-glutathionylation of overexpressed (upper) and endogenous (lower) PFKFB3. HeLa cells were treated with the indicated doses of BCNU to induce S-glutathionylation and the experimental methods were the same as (b) and (c). Immunoblotting shows PFKFB3 levels before (input, bottom) and after precipitation (upper).