Journal: The Journal of Biological Chemistry

Article Title: Ectopic Mineralization in the Middle Ear and Chronic Otitis Media with Effusion Caused by RPL38 Deficiency in the Tail-short ( Ts ) Mouse *

doi: 10.1074/jbc.M110.184598

Figure Lengend Snippet: Molecular genetics of Ts. A, genetic map of the Ts interval on distal MMU 11. Shown is the genetic linkage map obtained from the F2 intercross at the distal portion of mouse chromosome 11 (MMU 11), where locations of genetic markers are shown on the right, their genetic distance in centimorgans shown on the left, followed by the number of recombinants per meioses that separate each marker. The nonrecombinant critical Ts region is shown in red. B, insertion/deletion PCR screen. A single forward primer (F) and five progressively more distal located reverse primers (R31, -32, -34, -36, and -38) were predicted to produce products ranging from ∼19.5 to ∼21.0 kb. Instead, products ranging from 1.5 to 3.0 kb were observed exclusively from Ts/+ DNA template, indicating the absence of ∼18 kb of genomic DNA on the Ts chromosome. Shown is a SYBR Green-stained 2% agarose gel separating the amplified products. Lane M, DNA size standard. C, physical map of the Ts critical region. From top to bottom is shown the genetic region on chromosome 11 defined by markers D11Ntra3 and D11Ntra5 with their physical location given at the top in kb. Filled ovals represent the approximate locations of recombination events, and the red box indicates the location of the Ts critical interval. The arrow marks the position and orientation of Rpl38 (ribosomal protein L38), Ttyh2 (tweety homolog 2), and Dnaic2 (dynein, axonemal, intermediate chain 2). Sequencing chromatogram of the PCR fragment amplified from the Ts chromosome. Bases shaded in red represent the insertion sequence. D, verification/genotyping PCR scheme. PCR primers flanking the Ts mutation (a and b), within the deleted region (a′ and b′), and within the insertion (c and d) were generated. All +/+ DNAs tested (n = 9) yielded only fragments a + a′ and b + b′, whereas all Ts/+ DNAs tested (n = 13) yielded a + b, a + a′, b + b′, a + c, and d + b.

Article Snippet: Protein lysates of HEK293T cells transfected with and without Myc-tagged human Rpl38 ( {"type":"entrez-protein","attrs":{"text":"NP_001030335","term_id":"78214522","term_text":"NP_001030335"}} NP_001030335 ) were obtained from OriGene.

Techniques: Marker, SYBR Green Assay, Staining, Agarose Gel Electrophoresis, Amplification, Sequencing, Mutagenesis, Generated

Journal: The Journal of Biological Chemistry

Article Title: Ectopic Mineralization in the Middle Ear and Chronic Otitis Media with Effusion Caused by RPL38 Deficiency in the Tail-short ( Ts ) Mouse *

doi: 10.1074/jbc.M110.184598

Figure Lengend Snippet: Tissue expression and immunolocalization of Rpl38. A–C, Western blots of protein extracts from HEK293T cells (A) and tissue extracts of adult +/+ and Ts/+ mice (B) in the presence (+) and absence (−) of Pb863-specific peptide (C). Left side of each panel indicates the molecular mass in kDa. A, membranes stained with Pb863, Pb865, and anti-Myc antibody of HEK293T cells transfected and nontransfected with a mouse RPL38-myc fusion construct. Black dot indicates Rpl38-specific band present in the transfected but not in untransfected lysate. B, Western blot of protein extract from the ear and blood of +/+ and Ts/+ mice blotted with Pb865 and β-actin antibody. Arrowhead indicates the ∼8-kDa Rpl38 protein band. Arrows point to higher molecular weight bands in ear lysate. Protein lysates were also hybridized with β-actin antibody to control for equal loading. C, ear and blood protein lysates were incubated with Pb865 in the presence (+) and (−) absence of Pb865-specific peptide. Note that both the ∼8-kDa and higher molecular weight protein species are effectively blocked by the peptide. Membranes were hybridized with β-actin antibody to control for equal transfer efficiency. D--N, shown are confocal images of cochlear coil (D and E) and stria vascularis (F) whole mount preparations, cryosections of organ of Corti (G), stria vascularis (H), otic capsule (I), and blood spreads (J and K) stained with the Pb865 antibody (red channel). E–I and L--N, counterstained with DAPI (blue channel); E and F, counterstained with phalloidin (green channel); L, staining with nonspecific rabbit IgG antibody. J, inset shows high magnification of Z-stack of Rpl38-positive erythrocyte showing staining near the plasma membrane. White arrows (D–N) point to Rpl38-positive staining, and arrowhead in L points to nonstaining erythrocytes. M and N, staining of stria vascularis (M) and blood spread (N) with Pb865 in the absence (−) and presence (+) of Pb865-specific peptide. Note that Pb865 staining is blocked in the presence of competing peptide. pe, periosteum; eb, endochondral bone; el, endosteal layer; sl, spiral ligament; MEC, middle ear cavity. Scale bars, E and F, 10 μm; G and H, 20 μm; I, 50 μm; J, K and N, 5 μm; L and M, 20 μm.

Article Snippet: Protein lysates of HEK293T cells transfected with and without Myc-tagged human Rpl38 ( {"type":"entrez-protein","attrs":{"text":"NP_001030335","term_id":"78214522","term_text":"NP_001030335"}} NP_001030335 ) were obtained from OriGene.

Techniques: Expressing, Western Blot, Staining, Transfection, Construct, Molecular Weight, Control, Incubation, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Ectopic Mineralization in the Middle Ear and Chronic Otitis Media with Effusion Caused by RPL38 Deficiency in the Tail-short ( Ts ) Mouse *

doi: 10.1074/jbc.M110.184598

Figure Lengend Snippet: Transgenic rescue of Rpl38 deficiency. A, number of mice with a Ts/+ genotype as function of normal and short tail in the presence (+) or absence (−) of the transgene (tg). Right panel shows representative x-ray photographs of the tail morphology in transgene-positive (left) and transgene-negative (right) Ts/+ animal; scale bar, 1 cm. B, DPOAE emission levels in db SPL relative to noise floor of the 2f1 − f2 frequency at f2 = 16 kHz and L1 = 55 db SPL input; bars are mean ± S.E.; ***, p < 0.001; n.s. = not significant. C, ABR thresholds in db SPL at click stimulus; bars are mean ± S.D.; **, p < 0.01; n.s. = not significant.

Article Snippet: Protein lysates of HEK293T cells transfected with and without Myc-tagged human Rpl38 ( {"type":"entrez-protein","attrs":{"text":"NP_001030335","term_id":"78214522","term_text":"NP_001030335"}} NP_001030335 ) were obtained from OriGene.

Techniques: Transgenic Assay

Journal: The Journal of Biological Chemistry

Article Title: Ectopic Mineralization in the Middle Ear and Chronic Otitis Media with Effusion Caused by RPL38 Deficiency in the Tail-short ( Ts ) Mouse *

doi: 10.1074/jbc.M110.184598

Figure Lengend Snippet: Skeletal and auditory phenotypes in Rpl38 transgenic Ts mice Number of tails and ears that express the indicated phenotype are given as mean ± S.D. p , ANOVA test. Ts , Tail-short; tg, transgene.

Article Snippet: Protein lysates of HEK293T cells transfected with and without Myc-tagged human Rpl38 ( {"type":"entrez-protein","attrs":{"text":"NP_001030335","term_id":"78214522","term_text":"NP_001030335"}} NP_001030335 ) were obtained from OriGene.

Techniques: Transgenic Assay