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. 2011 Oct 12;6(10):e26034. doi: 10.1371/journal.pone.0026034

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Hiroe et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Histological phenotypes of the lung of GAK-kd^+/+ and GAK-kd^-/- pups. Sections of their lungs were stained with hematoxylin and eosin. Bar = 50 µm. (B and C) Total number (B) and total area (C) of alveolar compartments were counted in the images of four GAK-kd^+/+ pups and five GAK-kd^-/- pups. The average values of the bars were calculated in 4 independent areas under a microscope for each pup using Image J (version 1.44) public domain Java image processing package (National Institute of Health, Bethesda, Maryland, USA). Standard deviations are shown as error bars, which were calculated using excel 2003 software. The average values of the bars (blue and yellow) were calculated from average values of four GAK-kd^+/+ and five GAK-kd^-/- pups, and the values were statistically significant (**; P<0.01, *; P<0.05). Error bars indicate standard deviations (Student's t-test is used throughout). (D) Comparison of the thickness of the alveolar septum (µm) measured on the images of two independent areas (a and b) of four GAK-kd^+/+ and five GAK-kd^-/- pups. The results were statistically significant (*; P<0.05). (E) Western blotting using antibodies against EGFR, SP-A, E-cadherin and GAPDH (loading control) of lung extracts of GAK-kd^+/+ and GAK-kd^-/- pups. (F) Immunostaining of the lungs of GAK-kd^+/+ and GAK-kd^-/- pups with antibodies against SP-A, E-cadherin, EGFR-pS1047 and EGFR-pT654. The images show the enlarged views of the regions indicated by squares in Figure S5. Images were recorded using a microscope (BX51, Olympus) equipped with a CCD camera (DP72, Olympus). Bar = 50 µm.