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. 2016 Nov 15;27(23):3705–3716. doi: 10.1091/mbc.E16-05-0318

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© 2016 Zhang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — Expression and localization of sperm flagellar proteins in control and Ift20 mutant mice. (A) Testicular expression levels of a major component of sperm tail (ODFs), ODF2, and a central apparatus protein, SPAG16L, are not changed in the Ift20 mutant mice. Left, representative Western blot results using specific antibodies to examine testicular ODF2 and SPAG16L expression levels in control and Ift20 mutant mice. β-Actin and β-tubulin were used as controls. Right, quantitation of relative ODF2 and SPAG16L expression from six control and six Ift20 mutant mice normalized by β-actin and β-tubulin. There is no significant difference in expression levels between control and mutant mice. (B) ODF2 and SPAG16L are not assembled into flagella correctly in Ift20 mutant mice. Representative image of localization of ODF2 in the elongating spermatids of a control mouse (left) and an Ift20 mutant mouse (right). Specific signal (red) was detected in the developing tail of an elongating spermatid of the control mouse. However, ODF2 signal was still in the cytoplasm of an elongating spermatid of Ift20 mutant mouse. (C) Representative image of localization of SPAG16L in the elongating spermatids of a control mouse (left) and an Ift20 mutant mouse (right). Specific signal (red) was detected in the developing tail of an elongating spermatid of the control mouse. However, SPAG16L was still in the cytoplasm of an elongating spermatid of Ift20 mutant mouse. (D) Representative image of localization of ODF2 in the epididymis sperm of a control mouse (left) and an Ift20 mutant mouse (right). Specific signal (red) was observed in the principal piece of the control mouse. However, no specific signal was seen in Ift20 mutant mouse. (E) Representative image of localization of SPAG16L in the epididymis sperm of a control mouse (left) and an Ift20 mutant mouse (right). Specific signal (red) was detected in whole sperm tail of the control mouse. However, SPAG16L signal was not observed in the few developed sperm of the Ift20 mutant mouse. (F) The distribution of testicular ODF2 and SPAG16L in sucrose gradient is altered in Ift20 mutant mice. Note that both ODF2 and SPAG16L had a wider distribution in the gradients. However, distribution of the two proteins appeared to shift to the lighter fractions, indicating that the two proteins were assembled into the complexes with light density in the absence of IFT20.