DHODH activity in Jurkat cells
Intracellular DHODH enzymatic activities was analyzed as described
before(Yin, Kabashima, Zhu, Shibata & Kai, 2017). Jurkat T cells were
cultured in RPMI-1640 with 10% FBS and treated with different
concentrations of PIP. After 24 h incubation, cells were harvested by
centrifugation and lysed in NP-40 lysis buffer at 4°C for 15 min.
Lysates were clarified at 12000 rpm for 10 min and the subsequent
supernatant was used in DHODH reaction. Lysate
(200 μL) was incubated in buffer A
(the final volume,1.0 mL), containing 500 mM DHO, 200 mM
K2CO3-HCl
(pH 8.0), 0.2 Triton X-100, and 100 μM CoQ0 at 37 °C for 1 h. Mixture of
enzyme reaction (200 μL) was mixed with buffer B, containing 1 mM
4-TFMBAO, 2 mM K3[Fe(CN)6], and 20 mM
K2CO3 and then heated at 80 °C for 5
min. The reaction was stopped by cooling in ice and the fluorescence
signal was recorded using a PT1-QM4 steady-stead fluorimeter (PTI, USA)
at excitation and emission wavelengths of 340 and 460 nm, respectively.