G6pd activity colorimetric assay kit

G6PD was purified using a HisTALON Gravity Column Purification kit (TaKaRa Clontech). As a starting material, we used 10 g of cell pellet. The pellet was suspended in 5 mL of lysis buffer and incubated on ice for 15 min. The suspension was sonicated on ice with 10 short pulses (10 s) followed by pauses (30 s) and was centrifuged at 10,000 × g for 20 min at 4°C. The supernatants were collected and loaded onto the column, which had been equilibrated in washing buffer. The eluate was collected in 1 mL microcentrifuge tubes. After several washes, the proteins were eluted using 8 mL of elution buffer (containing 150 mM imidazole). Enzyme activity was measured using a G6PD activity colorimetric assay kit (BioVision, catalog No. K757). The amount of NADH that was generated between time points was quantified at OD_450nm. One unit (U) of G6PD activity was defined as the amount of enzyme that catalyzes the conversion of 1.0 μmol of glucose-6-phosphate to 6-phosphoglucono-δ-lactone and 1.0 μmol of NAD⁺ to NADH per min at 37°C.

Cells were seeded in 12-well plates, treated with inhibitors, and then stimulated with control or estradiol (10 nM). Cells (1 × 10⁵) were collected and lysates were extracted with 200 μl of extraction buffer following the manufacture's protocol. G6PD activity was quantified using G6PD Activity Colorimetric Assay kit (Biovision, Milpitas, CA, USA). G6PD activity was normalized to total protein levels.