Figure 1: Inactivation of GSK3β by p38 MAPK is specifically induced by DSBs. | Nature Communications

Figure 1: Inactivation of GSK3β by p38 MAPK is specifically induced by DSBs.

From: Inactivation of nuclear GSK3β by Ser389 phosphorylation promotes lymphocyte fitness during DNA double-strand break response

Figure 1

(a) Western blot analysis of P-S389 GSK3β, P-S9 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK in total thymocytes (T), double positive thymocytes (DP), double negative thymocytes (DN), mature CD4 T cells and mature CD8 T cells. Actin is shown as a loading control. (b) Mouse CD4 splenocytes were treated with media alone (Med) or stimulated with anti-CD3 and anti-CD28 Abs for 18 h and the levels of P-S389 GSK3β, GSK3β, P-p38 MAPK and p38 MAPK were determined by western blotting. Thymocytes are included as a positive control. (c) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h and the levels of P-S389 GSK3β, total GSK3β, P-p38 MAPK and total p38 MAPK were determined by western blotting. (d) Mouse CD4 splenocytes were treated with media alone (Med) or doxorubicin (Dox) for 18 h. Levels of P-S389 GSK3β, total GSK3β and γH2AX were determined by western blotting. GAPDH is shown as a loading control. (e) Mouse CD4 splenocytes were treated with doxorubicin for 18 h and cell death was measured by AnnexinV staining and flow cytometry (n=3, ±s.e.m.). (f) Human CD4 cells were treated with media alone (Med) or doxorubicin (Dox) for 18 h. P-T390 GSK3β, total GSK3β and γH2AX were examined by western blotting. (g) WT and T-cell-specific p38α conditional knockout (cKO) mice were left unexposed or exposed to 4 Gy of X-rays. After 1.5 h, CD4 splenocytes were isolated and P-S389 GSK3β and total GSK3β assessed by western blotting. Actin is shown as a loading control. (h) Relative levels of total GSK3β, P-T390 GSK3β and the relative ratio of P-T390 GSK3β to total GSK3β at baseline (BL) and after radiotherapy (Rad) in CD4 cells from breast cancer patients, determined by the Odyssey system (n=4). (i) Relative ratio of P-T390 GSK3β to total GSK3β in CD4 cells from healthy donors isolated in two different days (baseline 1 and 2) for each subject (n=3). *P value<0.05 as determined by paired t-test. (a–g) Data are representative of three or more independent experiments.

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