Oxidative Stress Produced by Hyperthyroidism Status Induces the Antioxidant Enzyme Transcription through the Activation of the Nrf-2 Factor in Lymphoid Tissues of Balb/c Mice

Figure 6

Activation of Nrf-2 mediated by PKC and ERK kinases. (a) Lymph nodes and spleen cells of euthyroid and hyperthyroid mice were incubated in the absence or presence of staurosporine (5 nM) for 2 hours, and then, the PKC activity was evaluated as described in Materials and Methods. The results shown are representative of 4 independent experiments performed in triplicate. differs significantly from the lymphoid cells of euthyroid mice without treatment with . ^#Cells treated with staurosporine differ significantly from their respective controls with . (b) The protein expression of PKCα (76.8 kDa), PKCβ (76.7 kDa), and PKCγ (78.4 kDa) was determined by western blot assays from cell extracts obtained from lymph nodes and spleens of euthyroid and hyperthyroid mice. Representative immunoblots from 4 independent experiments are shown. The bar graph shows the densitometric analysis of each band relativized to β-tubulin used as housekeeping. The smooth bars correspond to the lymph node samples, and the stripe bars correspond to the spleen samples. differs significantly from the lymphoid cells of euthyroid mice with . (c) Lymph nodes and spleen cells of euthyroid and hyperthyroid mice were incubated in the absence or presence of PD 98059 (20 μM) for 2 hours, and then, the phospho-ERK expression was evaluated by western blot assays from cell extracts obtained from these cells. Representative immunoblots from 3 independent experiments are shown. The bar graph shows the densitometric analysis of each band relativized to total ERK. differs significantly from the lymphoid cells of euthyroid mice without treatment with . ^#Cells treated with PD 98059 differ significantly from their respective controls with . (c) Phosphorylation of Nrf-2 was analyzed by western blot assays from protein extracts obtained from lymphoid tissue cells of hyperthyroid mice, preincubated in the absence or presence of staurosporine (5 nM) or PD 98059 (20 μM) or both inhibitors together. Representative immunoblots from 3 independent experiments are shown. The bar graph shows the densitometric analysis of each band relativized to β-tubulin used as housekeeping. differs significantly from the lymphoid cells of euthyroid mice without treatment with . ^#Cells treated with PKC or ERK inhibitors differ significantly from lymphoid cells of hyperthyroid mice without treatment with .

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