lis1 protein  (Thermo Fisher)

Journal: PLoS Biology

Article Title: Mouse screen reveals multiple new genes underlying mouse and human hearing loss

doi: 10.1371/journal.pbio.3000194

Figure Lengend Snippet: ABR thresholds were significantly raised at 8 weeks old in mutants (A,E) (Mann–Whitney U test, p < 0.001) but were highly variable between individuals (grey lines), while click-evoked ABR waveforms were close to normal (B,F show group mean waveforms at 20 dB above threshold), and reduced but nonsignificant amplitude growth with increasing stimulus level in mutants (C,D,G,H) (Mann-Whitney-Wilcoxon test with Holm correction for multiple testing). Ywhae homozygotes (red) and wild-type littermates (green) with individual mutant thresholds (grey) on a 50% C57BL/6N, 50% 129S5 (A-D) ( n = 18 homozygotes, 12 wild types for thresholds; n = 10 homozygotes, 11 wild types for amplitude functions) or a 12.5% C57BL/6N, 87.5% 129S5 genetic background (E-H) ( n = 9 homozygotes, 13 wild types for thresholds; n = 6 homozygotes, 13 wild types for amplitude functions). Heterozygotes have normal ABR thresholds. (I-L) Coronal sections of the middle ear in wild type (I,J) and homozygous Ywhae mutants (K,L). The tympanic membrane is retracted in the mutant, and the middle ear contains inflammatory debris. The mucosa is thicker in the mutant (double-headed red arrow), containing granulation tissue (‘G’), infiltrated immune cells (black arrow in L), and foamy macrophages (red arrowhead). Black rectangles in I and K indicate the areas enlarged in J and L. Scale bar for I and K is 1 mm; in J and L it is 100 μm. n = 2 homozygotes, 2 heterozygotes, 4 wild types. M,N. Scanning electron micrographs of the middle ear epithelium near the opening of the Eustachian tube in wild-type (M) and homozygous Ywhae mutant (N), showing widespread goblet cells and fewer ciliated epithelial cells in the mutant compared with the wild type, which is rich in ciliated cells. Scale bar is 10 μm. n = 5 homozygotes, 5 heterozygotes, 3 wild types. O,P. Scanning electron micrographs of the organ of Corti in a heterozygote (O) and homozygous mutant (P) showing normal appearance. OHCs are shown at the top, each with a V-shaped stereocilia bundle, and IHCs at the bottom. Images taken from 60% of the distance along the cochlear duct from the base. n = 7 homozygotes, 5 heterozygotes, 2 wild types. Scale bar is 10 μm. Q. Design of the Ywhae tm1e(EUCOMM)Wtsi mutation with exons in grey, FRT sites in green, loxP sites in red, and lacZ and neo components of the inserted construct labelled. R. Western blot of Ywhae and Pafah1b1 (Lis1) protein in brain showing no detected Ywhae in homozygous mutants (−/−), while expression of Pafah1b1 (encoded by a nearby gene) was unaffected. Vinculin was used as a loading control. n = 2 homozygotes, 2 heterozygotes, 4 wild types. S. Body weight growth with age in males (left) and females (right), showing significantly reduced weights in homozygotes (red) compared with wild types (green) (males, n = 7 homozygotes, 6 wild types; females, n = 7 homozygotes, 7 wild types; mixed model framework test as described by Karp and colleagues at 4 weeks, 16 weeks, and area under the curve, p = 6.9 × 10 −3 , 0.013, and 6.9 × 10 −3 respectively). T-V. X-rays of wild type (T) and homozygotes (U) showing shorter skull length (V) in the mutants ( p = 3.9 × 10 −5 ; n = 6 male and 5 female homozygotes and 5 male and 6 female wild types; mixed model framework test, males and females p = 3.9 × 10 −5 ). All plots are means ± standard deviation. Plotted data points are given in . ABR, auditory brainstem response; Ck, click; EAC, external auditory canal; FRT, flippase recombinase target; IHC, inner hair cell; lacZ , gene encoding β-galactosidase; loxP, locus of crossover in P1 bacteriophage; M, malleus; MEC, middle ear cavity; OHC, outer hair cell; SL, sensation level; SPL, sound pressure level; TM, tympanic membrane.

Article Snippet: A primary rabbit polyclonal antibody directed against a peptide mapping within a divergent domain of human YWHAE was used to detect the protein (T-16, sc1020, Lot: C1914; 1:200, Santa Cruz Biotechnology, Dallas, TX; Antibody Registry AB_630821) [ ], PAFAH1b1/LIS1 was detected with recombinant rabbit monoclonal raised against a synthetic peptide corresponding to residues in human LISs1 (ab109630, Lot: GR55503-8; 1:750, Abcam, Cambridge, UK; Antibody Registry AB_10861275), and a blot with an additional LIS1/PAFAH1b1 rabbit polyclonal antibody generated against synthetic peptide corresponding to residues surrounding Gly298 of human LIS1 protein was used for validation (12453S Lot:1, Cell Signaling Technology, Danvers, MA).

Techniques: MANN-WHITNEY, Mutagenesis, Membrane, Construct, Western Blot, Expressing, Control, Standard Deviation