Fig. 3.

Inactivation of the IKKβ pathway reduces GCLC and GCLM expression. Wild-type, Ikkβ(−/−), Tnfr1(−/−), and Traf2(−/−) cells were labeled with CM-H2DCFDA under normal growth conditions and were analyzed by Flow cytometry (A), and used for measurement of H₂O₂ release by catalase-inhibited luminol chemiluminescence (B). The time-courses for H₂O₂ release are shown in the line plots as luminescence units (LU)/μg cell protein, with the insert depicting the area under each curve (AUC) as nanomoles of H₂O₂ release per milligram of protein. Each value represents the average of two experiments. Total RNA was isolated from wild-type and Ikkβ(−/−) cells (C), and wild-type cells were treated for 24 h with 0.1% DMSO, 10 μM JSH23, 1 μM BMS-345541, and 0.5 μM TPCA-1 (E). The RNA were subjected to reverse transcription followed by quantitative RT-PCR. Results showed the levels of Gclc/Gapdh and Gclm/Gapdh in Ikkβ(−/−) relative to those in wild-type cells designated as 1. D, total cell lysates from wild-type, Ikkβ(−/−), and Ikkβ(−/−) cells infected with IKKβ adenovirus were subjected to Western blotting. The GCLC/β-actin and GCLM/β-actin levels were compared with those in wild-type cells designated as 1. F, enzyme activities in Ikkβ(−/−) cells were compared with those in wild-type cells, designated as 100%. All results are presented as the mean values ± S.E. from at least three independent experiments. Statistical analyses were done compared with the mean values in control wild-type cells. **, p < 0.01; ***, p < 0.001 were considered significant.