Figure 4

(a) Immunoblot analysis of nuclear extracts from differentiated SK-N-SH cells exposed to 1–4 h OGD. RelA Lys310 acetylation significantly increased after 4 h OGD. (b) Neuronal SK-N-SH cells were transfected with wild-type RelA and RelA-K310R plasmids or with a pSG5 empty vector for 24 h and then exposed to OGD for 4 h. The OGD-induced RelA acetylation on Lys310 was significantly reduced in RelA-K310R expressing cells. (c) Data from densitometric analysis of RelA and RelA Ac-K310 immunoblots are expressed as ratios to relative nucleolin levels. Bars are the means±S.E.M. of three separate experiments; ^*P<0.05 versus corresponding control value. (d) Cell survival was measured in SK-N-SH cells exposed to 15 h OGD. RelA overexpression significantly enhanced OGD toxicity, whereas RelA Ac-K310R overexpression prevented cell loss. Bars are the means±S.E.M. of three separate experiments; ^*P<0.01 versus OGD in pSG5-expressing cells. (e) Primary cortical neurons were transfected with Bim luciferase reporter plasmid together with wild-type RelA or RelA-K310R plasmids or pSG5 empty vector for 24 h and then exposed to 3 h OGD. Luciferase activity was measured after 4 h. RelA overexpression enhanced OGD-induced Bim promoter activity, whereas RelA-K310R overexpression totally inhibited such activity. Bars are the means±S.E.M. of three experiments run in triplicate; *P<0.01, **P<0.05 versus OGD value in pSG5-expressing neurons