Journal: International journal of molecular sciences

Article Title: Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction.

doi: 10.3390/ijms24043924

Figure Lengend Snippet: Figure 4. Surface GluA2 AMPA receptor in neonatal hippocampal neurons is decreased in A350V neurons and is increased after heat treatment. (A) Representative images (20× magnification) of total (T.GluA2) and surface (S.GluA2) GluA2 AMPA receptor in wild-type (WT) and mutant (A350V) IQSEC2 hippocampal neurons. Neurons treated with heat (40 ◦C) for 1 h are labelled as MUT.H.S or WT.H.S. (B) Quantification of relative surface GluA2 expression (ratio: S.GluA2/T.GluA2). Data are reported as the mean ± SEM obtained from at least 4 neuronal culture preparations for each of the 4 groups. ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: After four washes with PBS, cells were permeabilized and intracellular GluA2 labeled with 0.3% Triton X-100, 3% normal goat serum in PBS and 1% rabbit polyclonal intracellular anti-C-terminal GluA2 antibody (Synaptic System, cat # 182103) for 16 h at 4 ◦C.

Techniques: Mutagenesis, Expressing

Journal: Molecular Biology and Evolution

Article Title: Accelerated Evolution Analysis Uncovers PKNOX2 as a Key Transcription Factor in the Mammalian Cochlea

doi: 10.1093/molbev/msad128

Figure Lengend Snippet: Pknox2 mutants display hearing impairment. Hearing assessment of Pknox2 mutant mice ABRs ( A ) and DPOAEs ( B ) threshold measurements in 2-month-old Pknox2 +/+ and Pknox2 −/− mice at different frequencies (from 5.6 to 45.25 kHz). ( C ) ABR peak I amplitude at 80 dB. Statistical analysis: nonparametric Mann–Whitney test; * P < 0.05, ** P < 0.01, and *** P < 0.001. ( D ) Representative confocal images of IHC synapses from the basal turn of the cochleae immunolabeled for presynaptic ribbons (CtBP2-red) and postsynaptic receptor patches (GluA2-green) in Pknox2 −/− and Pknox2 +/+ mice. AntiCtBP2 antibody also weakly stains IHC nuclei. Scale bar, 7 µ m. ( E – G ) Puncta per IHC. Quantitative data obtained from Pknox +/+ and Pknox2 −/− mice. For each IHC, we analyzed the number of CtBP2 puncta ( E ), postsynaptic GluA2 receptor patches ( F ), and putative ribbon synapses ( G ). In Pknox2 −/− mice, an increase in the number of CtBP2 puncta, GluA2 receptor patches, and synapses on the basal region is observed ( Pknox2 +/+ n = 105 IHCs at the apical, 126 IHCs at the medial, and 139 IHCs at the basal from three animals; Pknox2 −/− n = 130 IHCs at the apical, 153 IHCs at the medial, and 137 IHCs at the basal region from five animals).

Article Snippet: The primary antibodies used in this study were: 1) goat anti-prestin antibody (Santa Cruz Biotechnology Inc. sc22692; 1:700); 2) anti-C-terminal binding protein2 (mouse anti-CtBP2 IgG1; catalog #612044, BD Biosciences; RRID:AB_399431; 1:200) to label the presynaptic ribbon; 3) anti-glutamate receptor 2 (mouse anti-GluA2 IgG2a; 1:2,000; MAB397, Millipore; RRID:AB_11212990) to label the postsynaptic receptor plaques; 4) rabbit anti-PKNOX1 (#PA5-66065, Thermo Fisher; 1:25); 5) rabbit anti-PKNOX2 (#PA5-65946, Thermo Fisher; 1:50); 6) mouse anti-Myosin VIIa (#E3018, Santa Cruz; 1:50); and 7) chicken anti-neurofilament heavy chain (#AB5539, Millipore, 1:1,000).

Techniques: Mutagenesis, MANN-WHITNEY, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss

doi: 10.3390/ijms22010003

Figure Lengend Snippet: ( A ) Expression of Neurabin I and II isoforms in the rat cochlea. PCR products (shown in duplicates) correspond to the neurabin I (520 bp) and neurabin II (561 bp) isoforms, respectively. In the absence of reverse transcriptase (-RT controls) no amplification was observed. ( B ) The most prominent neurabin I immunostaining was observed in sensory inner hair cells (IHC) and outer hair cells (OHC) in the organ of Corti (o/C) and spiral ganglion neurons (SGN). ( C ) The RGS4 antibody also demonstrated predilection for sensory hair cells (IHC and OHC) and SGN. RGS4 immunofluorescence was also observed in the auditory nerve fibres in the osseous spiral lamina (OSL) and blood vessels in the spiral limbus (SL). ( D ) Control section where the primary antibody was replaced by IgG isotype control. Abbreviations: DC, Deiters’ cells; RM, Reissner’s membrane; SV, stria vascularis. Scale bars, 50 μM.

Article Snippet: Whole mount tissues were then incubated overnight at RT with the following primary antibodies: rabbit polyclonal anti-Myosin-VIIa (Proteus Biosciences, 1:500), mouse anti-C-terminal binding protein 2 (CtBP2; IgG1; BD Biosciences, 1:500) and mouse anti-Glutamate receptor 2 (GluA2; IgG2; Merck Millipore, 1:500) in antibody solution containing 0.1% Triton X-100 in 0.1 M PBS with 5% NGS.

Techniques: Expressing, Amplification, Immunostaining, Immunofluorescence