Figure 4.

p38 MAPK mediates degradation of endogenous MafA under oxidative stress. A, Inhibitor of p38 MAPK, SB203580, rescues oxidative stress-mediated degradation of endogenous MafA in isolated pancreatic islets. Adult rat islets were cultured for the last 5 hours in the presence of DMSO or 20 μM SB203580 (SB20) in the presence or absence of CHX and 100 μM tBHP. Whole-cell extracts were subjected to immunoblots with α-MafA and α-actin antibodies. An image of a representative gel from 3 independent experiments is shown. Immunoblot of extracts from a second independent islet preparation for the effects of tBHP and SB20 is also shown. Graph on the right presents quantification of MafA band intensity normalized to the loading control (actin) from 3 independent experiments. Results are presented relative to the expression of MafA in CHX+DMSO as 1 ± SEM both in the presence and absence of tBHP. *, P < .01. B, p38 MAPK mediates degradation of MafA in the presence of oxidative stress. MIN6 cells infected with either GFP or dominant-negative (DN)p38 MAPK adenoviruses cultured for 24 hours were split into 6-well plates and cultured for an additional 24 hours with the last 5 hours in the presence or absence of 100 μM tBHP. Whole-cell extracts were subjected to immunoblots with α-MafA and α-actin antibodies. An image of a representative gel from 3 independent experiments is shown. Immunoblot from a second independent set of extracts from MIN6 cells infected with AdDNp38 MAPK is also shown. Graph on the right presents quantification of band intensity of endogenous MafA from 3 independent experiments. The results are presented relative to the expression of MafA (normalized to the loading control, actin) in MIN6 cells infected with GFP adenovirus (AdGFP) and treated with DMSO in the absence of tBHP as 1 ± SEM. *, P < .01. MIN6 cells infected with DNp38 MAPK adenovirus (AdDNp38 MAPK) had no significant difference in MafA protein levels in the presence or absence of tBHP.