Figure 2. DAPK1 induces pS23 accumulation and nuclear translocation. A, cDAPK1 activates p53 reporter genes via a direct binding of cDAPK1DD to p53DM in HEK293 cells. Data are mean ± SEM; n = 5. *p < 0.01 (compared with the vector). B, The p53 protein unchanged in the presence of cDAPK1. HEK293 cells were transfected with 2 μg plasmids, as indicated. At 72 h after transfection, cell lysates (30 μg proteins) were prepared and probed with antibodies against p53 or GAPDH, as indicated. Similar results were observed in each of the four experiments. C, The cellular extracts from p53−/− cells coexpressing cDAPK1 with p53 or p53A23 are blotted with anti-pS23 or anti-p53 after treatment with control buffer (CTR) or 100 units of alkaline phosphatase (PPA). Similar results were observed in each of the five experiments. D, cDAPK1DD-p53DM binding induces pS23 accumulation. cDAPK1 in the presence of 5 μm of a membrane-permeable p53DM (Tat-p53DM) peptide or scrambled control (Tat-s-p53DM), or vehicle, or cDAPK1DDΔ alone was expressed in DAPK1−/− cortical neurons. At 72 h after the expression, the cellular proteins were prepared and blotted with antibodies against pS23, p53, DAPK1, or GAPDH, as indicated. The pS23 band intensities are expressed as the ratio of the respective p53 protein. Data are mean ± SEM; n = 5. *p < 0.01. E, Inhibition of ERK or Tau does not affect DAPK1–p53 interaction. Cultured cortical neurons (DIV10) were transfected with cDNA encoding wDAPK1, cDAPK1DDΔ, or cDAPK1 with a scrambled control small interference RNA (s-siRNA), siRNA that specifically targets to ERK (E-siRNA) or Tau (T-siRNA) using rAAV1/2 viral vector. At 72 h after transfection, cell lysates were prepared and blotted with antibodies, as indicated. Similar results were observed in each of the four experiments. F, The expression of exogenous p53 does not cause pS23 accumulation. Cultured cortical p53+/+ and p53−/− neurons (DIV10) were transfected with cDNA encoding p53 using a rAAV1/2 viral vector. At 72 h after transfection, cell lysates were prepared and blotted with antibodies against pS23, p53, or tubulin, as indicated. Similar results were observed in each of the four experiments. G, The pS23 undergoes nuclear translocation. The nuclear proteins were purified from p53+/+ cortical neurons expressing an empty vector, wDAPK1, cDAPK1, or cDAPK1DDΔ using individual rAAV1/2 viral vector. At 72 h after expression, cell lysates were prepared and probed with antibodies, as indicated. pS23 band intensity is expressed as the ratio of the respective p53 protein. Data are mean ± SEM; n = 4. *p < 0.01. H, The pS23 is expressed in the nucleus. The cultured cortical neurons (DIV10) were transfected with wDAPK1 or cDAPK1 using the individual rAAV1/2 virus vector. At 72 h after the transfection, the cultures were stained with antibodies against p53 (green), or pS23 (red) and DAPI (blue), as indicated. Similar results were seen in each of the five experiments. Scale bar, 50 μm. I, pS23 is colocalized with mitochondrial protein, COX-IV. The cultured cortical neurons (DIV10) were transfected with cDAPK1 using the rAAV1/2 viral vector. At 72 h after the transfection, the cultures were stained with antibodies against pS23 and COX-IV, respectively.