Journal: The Journal of Neuroscience

Article Title: Neuregulin1 Nuclear Signaling Influences Adult Neurogenesis and Regulates a Schizophrenia Susceptibility Gene Network within the Mouse Dentate Gyrus

doi: 10.1523/JNEUROSCI.0063-24.2024

Figure Lengend Snippet: Key resources table

Article Snippet: Recombinant mouse ErbB4/Her4 Fc chimera protein, CF , R&D Systems , Catalog #4387-ER-050.

Techniques: Affinity Purification, Virus, Recombinant, Protease Inhibitor, Sequencing, RNAscope, Variant Assay, Plasmid Preparation, Software

Journal: The Journal of Neuroscience

Article Title: Neuregulin1 Nuclear Signaling Influences Adult Neurogenesis and Regulates a Schizophrenia Susceptibility Gene Network within the Mouse Dentate Gyrus

doi: 10.1523/JNEUROSCI.0063-24.2024

Figure Lengend Snippet: The V 321 L substitution decreases nuclear back signaling. A , Immunoblot of triplicate nuclear fractions isolated from pooled cortical and hippocampal lysates. Nrg1 ICD was detected using Santa Cruz Biotechnology antibody sc-348. Histone H3 served as a nuclear loading control and Na+/K+ ATPase as a marker for the membrane fraction ( N = 3 mice/genotype). B , Immunoblot of two replicates of membrane fractions isolated from pooled cortical and hippocampal lysates. NRG1 ICD was detected using Santa Cruz Biotechnology antibody sc-348. Na+/K+ ATPase served as a marker for the membrane fraction; note the lack of the nuclear marker Histone H3 or cytoplasmic marker CyclophilinA indicating clean membrane preps. FL Nrg1 is indicated with a yellow arrowhead as “Nrg1-FL,” and the membrane-bound C-terminal fragment not cleaved by γ-secretase is indicated as “Nrg1 TM-CTF.” A positive control consisting of total lysate from N2A cells transfected with a Type III Nrg1 plasmid is shown in the lane labeled “C.” ( N = 2 mice/genotype.) C , Left, Hippocampal neurons from WT (dark blue) and V 321 L (light blue) neonatal pups (P4) were cultured for 17 d in vitro and were stimulated with either vehicle (Veh), 20 nM sERBB4 (sB4), or 20 nM sErbB4 after a 24 h pretreatment with 20 µM of the γ-secretase inhibitor DAPT (DAPT). Neurons were fixed and stained using an antibody directed against the Nrg1 ICD and counterstained with DAPI. Scale bar, 10 µm. Right, Quantification of nuclear clusters of Nrg1-ICD. Neurons from WT mice show increased nuclear ICD clusters in response to sB4 stimulation, which is counteracted by pretreatment with DAPT (DAPT). Neurons from V 321 L mice do not respond to sB4 stimulation ( N = 6–13 neurons, 3 platings/mouse, 3 mice/genotype; one-way ANOVA p values corrected for multiple comparisons using Tukey’s post hoc test; WT Veh vs WT sB4, p < 0.0001 (****); WT sB4 vs WT sB4 + DAPT, **** p < 0.0001; WT sB4 vs V 321 L Veh, **** p < 0.0001; WT sB4 vs V 321 L B4, **** p < 0.0001). All other comparisons are statistically not statistically significant. D , Cortical neurons from embryonic WT (dark blue) and V 321 L mice (light blue; E18.5) were cultured for DIV3 and were stimulated with soluble ErbB4 (sB4), PI3K inhibitor WM, γ-secretase inhibitor L-685,458 (L6), WM + B4, or L6 + B4. Neurons that underwent no drug treatments/sB4 stimulation are indicated as the control group (C). Neurons were fixed and axonal length was quantified. (Two-way ANOVA with Tukey’s post hoc correction, WT C vs WT B4, **** p = 0.0002; WT L6 vs WT L6 + B4, ** p = 0.0047; V 321 L C vs V 321 L B4, *** p = 0.001; V 321 L WM vs V 321 L WM + B4, p = 0.1; V 321 L L6 vs V 321 L L6 + B4, * p = 0.03.) N = 20–37 neurons per genotype per condition. ns, not significant. E , Treatment and conditions same as in D . Quantification is for dendritic length. (two-way ANOVA w/ Tukey’s post hoc correction: WT C vs WT B4, ** p = 0.002; WT WM vs WT WM + B4, **** p < 0.0001). N = 20–37 neurons per genotype per condition. ns, not significant.

Article Snippet: Recombinant mouse ErbB4/Her4 Fc chimera protein, CF , R&D Systems , Catalog #4387-ER-050.

Techniques: Western Blot, Isolation, Control, Marker, Membrane, Positive Control, Transfection, Plasmid Preparation, Labeling, Cell Culture, In Vitro, Staining

Journal: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: Immunofluorescent localization of GFP-fused TULP1 (green) and Calnexin (red) in WT and mutant TULP1 expressing cells. Nuclei were stained using DAPI (blue) (A) WT-TULP1 expressing cells displayed predominant plasma membrane and some cytoplasmic staining patterns. (B-E) In contrast all four mutant TULP1 expressing cell lines (D94Y, R420P, I459K and F491L) displayed punctate co-localization patterns with the ER-resident marker Calnexin (merge: yellow). Scale bar = 10 μM. Approximately 100 cells per transfection were counted. Experiments were repeated twice.

Article Snippet: Visualization of the ER in fixed cells was achieved by staining cells with anti-Calnexin antibodies (an ER resident protein) (Cell Signaling Technologies, Carlsbad, CA).

Techniques: Mutagenesis, Expressing, Staining, Clinical Proteomics, Membrane, Marker, Transfection

Journal: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: (A) Overview of the subcellular fractionation procedure used for the isolation of ER microsomes. (B) Western blot analysis of ER microsomes isolated from GFP-fused WT or mutant TULP1 expressing hTERT-RPE-1 cells. Antibodies against Calnexin and Calreticulin were used to determine the ER fractions; whereas antibodies against Golgin97 and COX IV were used to determine the presence of Golgi or mitochondria, respectively. Expression of mutant TULP1 proteins (D94Y, R420P, I459K and F491L) was retained within the ER. Total cell lysate from untransfected HEK293T or hTERT-RPE-1 cells were used as controls. Actin was used a protein loading control.

Article Snippet: Visualization of the ER in fixed cells was achieved by staining cells with anti-Calnexin antibodies (an ER resident protein) (Cell Signaling Technologies, Carlsbad, CA).

Techniques: Fractionation, Isolation, Western Blot, Mutagenesis, Expressing, Control

Journal: bioRxiv

Article Title: Oligomerization and cellular localization of SLC26A11

doi: 10.1101/2024.04.29.591613

Figure Lengend Snippet: A – F , Confocal images of HEK293T cells ( A, C and E ) and B-ICC Clone C cells ( B, D and F ) co-expressing SLC26A4/pendrin, SLC26A7 and SLC26A11 together with the lysosomal marker Lamp1 or the ER marker Calnexin. G and H , box plot of Manders coefficients of colocalization analysis between SLC26A4/pendrin, SLC26A7 and SLC26A11 with the corresponding intracellular marker (SLC26A11/Lamp1 HEK293T , n = 20; SLC26A11/Lamp1 CloneC , n = 20; SLC26A11/Calnexin HEK293T , n = 19; SLC26A11/Calnexin CloneC , n = 18); (SLC26A4/Lamp1 HEK293T , n = 20; SLC26A4/Lamp1 CloneC , n = 14; SLC26A4/Calnexin HEK293T , n = 18; SLC26A4/Calnexin CloneC , n = 15); (SLC26A7/Lamp1 HEK293T , n = 25; SLC26A7/Lamp1 CloneC , n = 23; SLC26A7/Calnexin HEK293T , n = 25; SLC26A7/Calnexin CloneC , n = 21); One-way ANOVA (Tukey’s HSD post hoc test). Data are presented as means ± S.D. with n being the number of co-transfected cells analyzed. In boxplots, boxes indicate the upper and lower quartiles, and whiskers the upper and lower 90 percentiles. Scale bars = 10 µm.

Article Snippet: We received the lysosomal marker Lamp1 as a gift from Walter Mothes, Addgene plasmid #1817 , and the ER marker Calnexin as gift from Michael Davidson ( http://n2t.net/addgene:55005 ; RRID: Addgene_55005).

Techniques: Expressing, Marker, Transfection