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Figure 7 | BMC Biology

Figure 7

From: The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6 +/-mouse model of epithelial wound-healing delay

Figure 7

ERK1/2 phosphorylation in wild-type and mutant cultures is required for early wound response. (A–D) Immunolabeling of phospho-ERK1/2, (A) 10 minutes after wounding in Pax6 +/+ cultures; (B) 30 minutes after wounding in Pax6 +/+ cultures and (C) 10 minutes after wounding in Pax6 +/- cultures. (D) Exposing Pax6 +/- cells to EGF 90 s before wounding elicited a large phospho-ERK1/2 response after 10 minutes. In all cases, * indicates the wounded region. Scale bars = 100 μm. (E) Western blots of protein lysate of Pax6 +/+ and Pax6 +/- cultures. The same blot was probed sequentially with antibodies against Pax6, phospho-ERK1/2 (P-ERK1/2), and β-actin. Left hand lane: Pax6 +/- cultures with 10 minute EGF exposure as described above. Middle lane: Pax6 +/- cultures with no EGF exposure. Right hand lane: control Pax6 +/+ cultures. Correcting for the volume of actin staining, phosphor-ERK1/2 staining in control heterozygous cultures was estimated at 80% of wild-type levels. Addition of EGF prior to wounding did not change Pax6 levels in Pax6 +/- cultures, but increased phospho-ERK1/2 staining to levels comparable with or greater than Pax6 +/+. All samples were taken from pools of at least six corneal epithelial cell cultures treated identically. (F) Rates of wound healing of wild-type and mutant cells with addition of 10 μM U0126 (an inhibitor of phosphor-ERK1/2 signaling), 0.1% DMSO, 15 minutes prior to wounding (U), or with addition of U0126 prior to wounding and replacement of drug in fresh medium every 2 hours (U*), or Pax6 +/+ and Pax6 +/- cultures with addition of 0.1% DMSO only. (*, p < 0.01 compared with wild-type control).

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