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. 2015 Dec 1;8(12):1555–1568. doi: 10.1242/dmm.023176

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 7. — The inner ears of P1 Map3k1^goya/goya mice exhibit increased levels of p38 phosphorylation. (A) Box plot of the ratio of phosphorylated p38 to total p38 in goya inner-ear total protein lysates from Map3k1^+/+, Map3k1^goya/+and Map3k1^goya/goya mice. Three ears (one each from separate mice) were pooled for each lysate (number of lysates: Map3k1^+/+n=8, Map3k1^goya/+ n=7, Map3k1^goya/goya, n=8). A trend of increased p38 phosphorylation is observed in Map3k1^goya/goya inner-ear lysates when compared to Map3k1^+/+ (P=0.282). Average Map3k1^goya/+ p38 phosphorylation levels are intermediate between Map3k1^+/+ and Map3k1^goya/goya. (B-E) Immunohistochemical analysis of P1 Map3k1^goya/goya cochlea confirms the increased level of p38 phosphorylation observed in the Peggy Western data. Panels Bi and iii show phosphorylated p38 expression in the mid turn of the cochlea of P1 Map3k1^+/+ (i) and Map3k1^goya/goya (iii) mice. Widespread nuclear expression is seen in both genotypes; however, the depth of staining is greatly increased in Map3k1^goya/goya. Some nuclei in spiral ligament, spiral limbus and basilar membrane in the Map3k1^+/+ organ of Corti remained unstained with this length of chromogenic exposure. These results were consistent for littermate controls (Map3k1^+/+ n=2, Map3k1^goya/goya n=3). (C) Immunoratio was used to quantify the percentage of nuclei stained in each mid turn image. This analysis showed that the Map3k1^goya/goya mice (n=3) had significantly increased numbers of positively stained cells when compared to Map3k1^+/+ (n=2). Bii and iv show increased positive staining for phosphorylated p38 in the spiral ganglion neurons of Map3k1^goya/goya (iv) mice compared to Map3k1^+/+ (ii). (D,E) Immunostaining of P1 organ of Corti using anti-phospho JNK and anti-phospho ERK1/2 antibodies, respectively. No differences can be seen between Map3k1^+/+ (Di,Ei) or Map3k1^goya/goya (Dii,Eii) mice, although, interestingly, expression of both proteins is mainly observed below the basal surface of inner and outer hair cells, in contrast to the widespread nuclear expression of phosphorylated p38.