Figure 8

AP-2α regulates GN-11 neuron migration via Axl. (a) Three AP-2α binding sites were identified in the regulatory region (-1000/+1, +1 is the transcription start site (TSS)) of the mouse Axl gene (site 1 = GCCCCAAGG; site 2 = GCCAGGGGC; site 3 = GCCCAGGGG) and verified by chromatin immunoprecipitation (ChIP) using two primers at the indicated positions. Chromatin from GN-11 cells was cross-linked to proteins, extracted and immunoprecipitated with either AP-2α Abs (C-18 or 3B5 or Geneka) or non-specific IgG (negative control). DNA was analysed by polymerase chain reaction (PCR), using primers flanking the AP-2α putative binding sites in the Axl promoter. Input: not immunoprecipitated DNA. The experiment was repeated three times and one representative experiment is shown. (b) Schematic representations of the mouse Axl promoter fragments cloned into the pGL3-basic vector are shown (left panel). HeLa cells were transiently transfected with the various constructs together with a Renilla normalization vector (pRL-TK) and 48 hours later, luciferase activity was measured and normalized against Renilla activity. The experiment was performed in triplicate and repeated two or three times. One representative experiment is shown (right panel). RLU = relative luciferase units. (c) The effect of Axl down-regulation on cell movement was assessed for lentivirus-infected GN-11 neurons expressing either pLKO.1scr (scrambled) or pLKO.1Axl1 or pLKO.1Axl2. Axl down-modulation was evaluated by quantitative real-time PCR (left panel) while transwell migration (right panel) was analysed by plating the cells in serum-free medium in the upper chamber and allowing the cells to migrate over 10% foetal calf serum (FCS) medium for 18 hours through a porous membrane. The area occupied by migrating cells is shown. The experiments were performed in triplicate and repeated twice. Representative experiments are shown. The differences were statistically significant as measured by a two-tailed Student's t-test (***, p < 0.05). (In (b) and (c) the bars represent ± standard deviations.)