Journal: iScience
Article Title: mTORC2 regulates auditory hair cell structure and function
doi: 10.1016/j.isci.2023.107687
Figure Lengend Snippet: KO confirmation of HC-RicKO mice (A) Hemizygous Myo15 - Cre +/− mice were crossed with homozygous Rictor fl/fl mice harboring LoxP sites upstream and downstream of exon 4 and 5, respectively. Forward (F) and reverse primers (R) were used to detect Cre-mediated recombination, resulting in a 280 bp PCR product after Cre-mediated recombination of the Rictor allele. (B) PCR using genomic DNA from dissected organ of Corti (OC), spiral ganglion (SG) or vestibular organ (vest; macular and cristae ampullaris organs pooled) tissue, separate for left (L) and right (R) inner ears. Genomic DNA from outer ear skin of both genotypes was used as negative control of Cre-mediated recombination. Genomic DNA from heart samples of tamoxifen-induced cardiomyocyte-specific Rictor KO mice was used as positive control (pos ctrl). No template control (NTC) contains all PCR reaction components including primers but no DNA. Successful Cre-mediated recombination of the Rictor allele was only found in OCs and vestibular organs of HC-RicKO mice. (C) Representative images (maximum intensity projections) of the medial cochlear turn from 4-week-old mice stained with an antibody against Akt-pSer473. Myosin7a antibody, phalloidin and nuclear DAPI staining visualize the hair cells. Scale bar for all figures = 20 μm. (D) Quantification of mean fluorescence intensity (MFI) in arbitrary units (a.u.) of the Akt-pSer473 signal intensity in the medial cochlear turn of 4-week-old mice. n = 3 mice per genotype. Results are presented as means ± SDs. Student’s t test, ∗p < 0.05.
Article Snippet: Mouse monoclonal anti-myosin7a IgG1 , Developmental Studies Hybridoma Bank , Cat#138-1s; RRID: AB_2282417.
Techniques: Negative Control, Positive Control, Staining, Fluorescence