Figure 9.

Phosphorylation of p53 at Ser15 induced by AMPA is JNK/CK2 dependent. Oligodendrocytes were treated for 30 min with 10 μM AMPA plus 100 μM CTZ, in absence or presence of JNK or CK2 inhibitors, and the different assays were carried out at the indicated times. (A) WB analysis of total p53 and p-p53(Ser15) at 30 and 60 min after excitotoxic stimulus, in absence or presence of 1 μM SP600125. Exposure to AMPA induced an increase in expression levels of both total p53 and p-p53(Ser15), which were abolished by the JNK-inhibitor SP600125. The level of JNK phosphorylation was used as a control of the effectiveness of the treatment and β-actin was the internal loading control. **p < 0.01, ***p < 0.001 (cells treated with AMPA respect to control cells); ^#p < 0.05, ^##p < 0.01 (cells treated with SP600125 respect to cells treated with only AMPA, at the same time). (B) AMPA triggered increases in p53 expression level, revealed with an antibody against total p53, which was reverted by JNK inhibitor SP600125 and CK2 inhibitor TBB. Scale bar: 10 μm. (C) Cells were treated with AMPA, as indicated above, in the presence of SP600125 or TBB and fixed at 30 min after stimulus. Immunofluorescence analysis using the specific antibody against p-p53(Ser15; green) showed that AMPA-induced activation of p53 through its phosphorylation in Ser15 was not detected in presence of JNK or CK2 inhibitors and neither its mitochondrial accumulation (revealed by COX-IV antibody; red). Scale bar: 10 μm.