Journal: Scientific Reports

Article Title: Live Neuron High-Content Screening Reveals Synaptotoxic Activity in Alzheimer Mouse Model Homogenates

doi: 10.1038/s41598-020-60118-y

Figure Lengend Snippet: Validation of PSD95-mVenus postsynaptic, VAMP2-mRFP presynaptic, and colocalized puncta. The majority of anti-VAMP2 and anti-Synapsin1 stained puncta were colocalized with endogenous VAMP2-mRFP puncta. Similarly, the majority of the antibody stained anti-PSD95 and anti-Homer2 puncta were colocalized with endogenous PSD95-mVenus puncta. ( a–c ) Presynaptic VAMP2-mRPF in singly transgenic mice was validated with immunocytochemistry stained ( a ) anti-VAMP2 and ( b ) anti-Syn1, ( c ) 92.1 ± 4.8% of stained VAMP2 puncta and 80.8 ± 3.9% of stained Syn1 puncta was colocalized with endogenous VAMP2-mRFP; ( d–f ) Postsynaptic PSD95-mVenus expression in singly transgenic mice was validated with immunocytochemistry stained ( d ) anti-PSD95 and ( e ) anti-Homer2, ( f ) 107.5 ± 4.5% of stained PSD95 puncta and 113.7 ± 6.2% of stained Homer2 puncta was colocalized with endogenous PSD95-mVenus (mean ± SD%, n = 3).

Article Snippet: All cells were blocked with 5% bovine serum albumin and 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 1 hour at room temperature, and then incubated with anti-Synaptobrevin 2, anti-Synapsin1, anti-PSD95, or anti-Homer2 (Synaptic Systems) antibodies at 1:500 in blocking solution overnight at 4 °C, subsequently washed in 0.1% Triton X-100 in PBS three times.

Techniques: Staining, Transgenic Assay, Immunocytochemistry, Expressing

Journal: Journal of proteomics

Article Title: A mass spectrometry-based proteomic analysis of Homer2-interacting proteins in the mouse brain

doi: 10.1016/j.jprot.2017.07.008

Figure Lengend Snippet: (A) Workflow of the Anti-Homer2 co-IP experiment. The different WT and KO fractions are colored in blue and red, respectively. (B) 2-D western blot images of a representative input fraction (top) and a representative co-IP sample (bottom) blotted for Homer2a and Homer2b proteins. (C) 1-D western blot image of fractions collected from a representative co-IP experiment performed using WT (left) and KO (right) mouse whole-brain samples and blotted for Homer2 proteins. (D) 1-D silver stained gel image of fractions collected from a representative co-IP experiment performed using WT (left) and KO (right) mouse whole-brain samples. Lanes in (A), (C), and (D) correspond to: (L), ladder; (1), Homogenate; (2), Pellet; (3), Supernatant (input fraction); (4), Pre-cleared supernatant; (5), Post-co-IP supernatant; and (6) co-IP sample. MW = molecular weight

Article Snippet: For the Homer2 co-IP verification experiment, whole brains were harvested from three male NIA C57BL/6 mice (3 months) purchased from Charles River Laboratories (Seattle, WA).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Staining, Molecular Weight

Journal: Journal of proteomics

Article Title: A mass spectrometry-based proteomic analysis of Homer2-interacting proteins in the mouse brain

doi: 10.1016/j.jprot.2017.07.008

Figure Lengend Snippet: (A) Of the 31 previously reported Homer2-interacting proteins, 11 were detected by MS1 full-scan filtering in the mouse whole-brain fractions used to co-IP Homer2 (yellow), 6 were detected in the co-IP samples (blue), and 14 proteins were not detected in either dataset (gray). (B) A total of 22 proteins were significantly enriched at a 10% FDR in WT co-IP samples. In addition to the four previously reported Homer2-interacting proteins (green), 18 novel proteins co-IPed with Homer2 (orange). FDR = false discovery rate.

Article Snippet: For the Homer2 co-IP verification experiment, whole brains were harvested from three male NIA C57BL/6 mice (3 months) purchased from Charles River Laboratories (Seattle, WA).

Techniques: Co-Immunoprecipitation Assay

Journal: Journal of proteomics

Article Title: A mass spectrometry-based proteomic analysis of Homer2-interacting proteins in the mouse brain

doi: 10.1016/j.jprot.2017.07.008

Figure Lengend Snippet: (A) Detection of previously reported Homer2-interacting proteins in the input fractions and co-IP samples. (B) Enrichment of previously reported and novel Homer2-interacting proteins in co-IP samples. Previously reported Homer2-interacting proteins that were detected in the input fractions and co-IP samples are colored yellow and blue, respectively. Previously reported and novel Homer2-interacting proteins that were significantly enriched in WT samples are colored green and orange, respectively. Table headers correspond to: (UniProt ID), mouse UniProt protein identifier; (Indistinguishable Group), proteins that contain peptides expressed in more than one gene product; (Gene) common gene name; (Protein Description) protein name; (Common Name) short protein name or alternative protein name; (FC Log2) Log2 transformed fold change; (FDR) false discovery rate (q ≤ 0.10) ; [SRM Verified (+2 peptides)], whether or not the protein was verified by SRM mass spectrometry using +2 charged peptides in a second co-IP experiment. N/A = not applicable.

Article Snippet: For the Homer2 co-IP verification experiment, whole brains were harvested from three male NIA C57BL/6 mice (3 months) purchased from Charles River Laboratories (Seattle, WA).

Techniques: Co-Immunoprecipitation Assay, Transformation Assay, Mass Spectrometry

Journal: Journal of proteomics

Article Title: A mass spectrometry-based proteomic analysis of Homer2-interacting proteins in the mouse brain

doi: 10.1016/j.jprot.2017.07.008

Figure Lengend Snippet: Cartoon depiction of a neuronal post-synapse. Previously reported Homer2-interacting proteins that were significantly enriched in WT samples are colored green and novel proteins are colored orange. The majority of Homer2-interacting proteins are NMDAR subunits or part of the NMDAR signaling pathway (gray area). Homer2 is not included as an interacting protein because our co-IP experiments cannot differentiate Homer2 proteins directly bound by the antibody from Homer2 homomultimers. Literature references for the involvement of these proteins in the NMDAR signaling pathway are included in Supplemental Table 3.

Article Snippet: For the Homer2 co-IP verification experiment, whole brains were harvested from three male NIA C57BL/6 mice (3 months) purchased from Charles River Laboratories (Seattle, WA).

Techniques: Co-Immunoprecipitation Assay

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A) The pedigree of a five-generation family segregating progressive ADNSHL. DNA samples were obtained for 9 unaffected and 10 affected individuals. The partially filled symbol represents a two-year-old female who carries the HOMER2 mutation but in whom a formal ABR has not been performed. Individuals who underwent TGE+MPS using either the deafness panel (OtoSCOPE) or WES are marked with an O or W, respectively. Genotypes of participating family members are shown below each symbol in single letter amino acid nomenclature: P for Proline and R for Arginine. ( B) Age-related typical audiograms (ARTA). Binaural mean air conduction thresholds (dB HL) are presented for the ages 10–70 years. Hearing levels ranged from 0 to 120 dB depending on age and frequency; the annual threshold deterioration (ATD) was 1.2–1.6 dB/year at all frequencies. ( C) Representative chromatograms from wild-type and mutant sequences. ( D) Diagram of HOMER2 structure; the amino acid numbering indicates the beginning and end of the EVH1 and CC domains. The CC includes the CDC42 binding domain (CBD), Leucine Zipper-A (LZA) and Leucine Zipper-B (LZB). The position of the p.Arg185Pro mutation is shown in red.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Mutagenesis, Binding Assay

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A ) Staining with F-actin shows three rows of OHCs and one row of IHCs in the cochlea. ( B ) Homer2 staining in the OHCs and IHCs shows localization to stereocilia. ( C ) Merged pictures showing co-localization of Homer2 with F-actin in HC stereocilia. ( D ) Zoomed view of OHCs shows pronounced localization of Homer2 to the tips of stereocilia. Scale bar represents 10μm.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Staining

Journal: PLoS Genetics

Article Title: HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

doi: 10.1371/journal.pgen.1005137

Figure Lengend Snippet: (A-D) Auditory brainstem responses (ABR) to broad band clicks (A) and tone bursts (B-D) at 8, 16, and 32 kHz in P14, P28 and P56 Homer2 -/- , Homer2 +/- and WT mice. (A) Raised mean ABR thresholds (dB SPL) were detected as early as P14 in Homer2 -/- mice and continued to increase with age. (B) At P14 Homer2 -/- mice had severe-to-profound hearing loss at high frequencies (16 kHz and 32 kHz) whereas hearing thresholds in their WT and Homer2 +/- littermates were in the normal range. (C) At P28 Homer2 -/- mice had hearing loss in the lower frequencies. (D) P56 Homer2 -/- mice had profound hearing loss across all tested frequencies (click, 8 kHz, 16 kHz and 32 kHz). (E-G) DPOAE levels. (E) In P14 Homer2 -/- mice, DPOAE amplitudes are significantly lower in the high frequencies (16, 22.6, and 32.0 kHz) as compared to their WT and Homer2 +/- littermates. (F) In P28 Homer2 -/- mice, DPOAE amplitudes are lower in nearly all frequencies (5.7, 8, 11.3, 16, 22.6, and 32.0 kHz). (G) In P56 Homer2 -/- mice, DPOAEs deteriorate across all frequencies consistent with profound hearing loss. (H) Representative Alexa-Fluor-488-phalloidin immunofluorescence shows no obvious hair cell death in cochleae in P56 Homer2 -/- (n = 5), Homer2 +/- (n = 4) and WT (n = 3) mice. Mean ABR thresholds and DPOAE amplitudes: Homer2 -/- mice are shown in red; Homer2 +/- in blue; and WT mice in green. The number of ears tested is shown in parentheses. P-values were calculated with One-way ANOVA and post-hoc T-test analysis. Asterisks indicate statistical significance: *P<0.05, **P<0.005, ***P<0.0005. Error bars represent SEM. Scale bar represents 10μm.

Article Snippet: Following infiltration using 0.3% Triton X-100 and blocking with 5% normal goat serum, we incubated the tissues in rabbit HOMER2 polyclonal primary antibody (NB100-98712, Novus Biologicals, Littleton, CO) diluted 1:1000 in PBS overnight at 4°C.

Techniques: Immunofluorescence