Fig 1: AKT regulates UVB-mediated nuclear translocation of TC45 in keratinocytesA. and B. Mouse primary keratinocytes were cultured and irradiated with UVB at three doses: 50, 100, or 150 mJ/cm2. Cells were collected at the indicated times after UVB exposure. Nuclear and cytoplasmic lysates were separately extracted. A. Western blot analysis of nuclear TC45 and phosphorylated STAT3 (pSTAT3) after UVB irradiation in keratinocytes. C: cytoplasmic fraction, no UVB irradiation. Lamin A/C was used as a control for nuclear fraction. B. Western blot analysis of cytoplasmic TC45 and pSTAT3 after UVB irradiation in keratinocytes. N: nuclear fraction, no UVB irradiation. LDH was used as a control for cytoplasmic fraction. C. Schematic representation of putative interaction motifs for 14-3-3 within the TC45 amino acid sequence identified by Scansite3 (http://scansite3.mit.edu). D. Western blot analysis of phosphorylated AKT (pAKT), AKT, phosphorylated AKT substrate, or 14-3-3s in response to UVB irradiation. 3PC keratinocytes were cultured and irradiated with UVB at a dose of 100 mJ/cm2. Cells were collected at the indicated times. E. Western blot analysis of nuclear TC45 in response to UVB irradiation. Cells were cultured and treated with AKT1/2 inhibitor (1 µM), Staurosporine (10 nM), Gö6983 (1 µM), RO-31-8220 (3 µM), or Gö6976 (1 µM) for 1 h before UVB irradiation (100 mJ/cm2, lanes 2-8). For activation of TC45 (lane 8), cells were treated with spermidine (0.2 µM), a known TC-PTP activator, for 1 h before UVB irradiation. Cells were then collected 3 h after UVB irradiation and nuclear lysates were isolated.