Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: ( A ) Workflow of quantitative proteomic analysis. Proteomic data were obtained from mice hippocampus, n =3. ( B ) Heatmaps of differentially expressed (DE) proteins in WT and ARIH1 +/- hippocampal samples. ( C ) Volcano plot for DE proteins (139 upregulated, 66 downregulated) in ARIH1 +/- hippocampal samples compared with WT hippocampus. Red and blue dots indicate statistical significance DE proteins. The hippocampal tissue of WT and ARIH1 +/- mice were dissected and whole lysis was prepared for immunoblot analysis ( D ) and total RNA was prepared for qPCR analysis ( E , F ) as described in the methods. The qPCR was probed by mouse ARIH1 or GIRK2 primers, n =4. The immunoblot was probed by anti-ARIH1 or anti-GIRK2 antibody. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls, n =4. * p < 0.05, ** p < 0.01, *** p < 0.001, ns . not significant, unpaired Student’s t test ( D , E , F ). Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: Lysis, Western Blot

Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: HT-22 cells were infected with lentivirus expressing scramble (NC) or Arih1-shRNA of two different on-target sequence (948, 949). NC and Arih1 knockdown cells were selected and maintained in cell culture media supplemented with puromycin (2.5μg/ml for selection, and 1μg/ml for maintaining) as described in the methods. ( A ) Whole cell lysates were prepared from HT-22 NC and Arih1 knockdown cells for immunoblot analysis probed by anti-Arih1 or anti-Girk2 antibodies. ( B ) Arih1 knockdown cells were transfected with vector or Flag-Arih1 plasmids. After 48hours transfection, whole cell lysates were prepared for immunoblot analysis probed by anti-Girk2, anti-Arih1, or anti-Flag antibodies. ( C ) HT-22 NC and Arih1 knockdown cells were transfected with Flag-Ub and HA-Girk2 plasmids. After 48hours transfection, whole cell lysates were prepared and immunoprecipitation was performed to examine the ubiquitination of Girk2 using anti-Flag or anti-HA antibodies. Showing blots are representative of at least 3 independent experiments. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls ( A 1 -A 2 , B 1 -B 4 ), or to corresponding input loadings ( C 1 ). ( D , E ) The total RNA was prepared from HT-22 NC and Arih1 knockdown cells for qPCR analysis with mouse ARIH1 or GIRK2 primers, n =5. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns . not significant, one-way ANOVA followed by Tukey’s test, n =3-6. Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: Infection, Expressing, shRNA, Sequencing, Knockdown, Cell Culture, Selection, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: bioRxiv

Article Title: ARIH1 deficiency impairs spatial learning and memory via GIRK2 upregulation in hippocampal CaMKII-expressing neurons in mice

doi: 10.1101/2025.03.10.625121

Figure Lengend Snippet: Schematic of the AAV-ARIH1 shRNA ( A ) and brain injection site ( B ) in mice. ( C ) The MWM experiment is composed of 3 sections which are adaptation (day 1), training (day 2 to 7), and testing (day 8). The latency on day 1 as well as the average latency (4 quadrants) on day 2 to 7 of AAV-Scramble or AAV-ARIH1 shRNA injected mice were calculated as described in the methods. During test on day 8, the latency ( D ) as well as times crossing the platform area ( E ) of AAV-Scramble or AAV-ARIH1 shRNA injected mice were recorded as described in the methods, n =10-12. ( F ) Representative image for showing the site of injection. Scale bar, 100μm. ( G ) The dorsal hippocampal tissue of AAV-Scr and AAV-ARIH1 shRNA injected mice were dissected and whole cell lysates were prepared for immunoblot analysis probed by anti-ARIH1 antibody. The mean intensity of bands was quantified using Image J and normalized to corresponding loading controls, n =3. ( H ) Representative image for showing co-expression of AAV-GFP with CaMKII, but not with GFAP or IB1. Scale bar, 20μm. ( I ) Representative image of immunostaining of Girk2 in mice with dorsal hippocampal injection of AAV-Scramble or AAV-ARIH1 shRNA. Scale bar, 100μm. ( J ) Quantification of the intensity of Girk2 staining, n =5. ( K ) The latency on day 1 as well as the average latency (4 quadrants) on day 2 to 7 of AAV-ARIH1 shRNA injected mice, administrated with Saline or Tipepidine ( i.p. , 20mg/kg), were calculated as described in the methods. During test on day 8, the latency ( L ) as well as times crossing the platform area ( M ) of AAV-ARIH1 shRNA injected mice, administrated with Saline or Tipepidine, were recorded as described in the methods, n =10-11. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired Student’s t test ( D , E , G , J , L , M ). * p < 0.05, two-way ANOVA followed by Bonferroni’s test ( C , K ). Mean ± SEM.

Article Snippet: The following antibodies were used: anti-ARIH1 (Ab3891, Abcam), anti-ARIH1 (NBP2-57888, Novus Biologicals), anti-GIRK2 (21647-1-AP, Proteintech), anti- GAPDH (60004-1-lg, Proteintech), anti-β-actin (66009-1-Ig, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-CaMKII (ab52476, Abcam), anti-GFAP (ab7260, Abcam), anti-Ib1 (011-27991, Wako), anti-Parvalbumin (ab32895, Abcam), and anti-Somatostatin (ab140665, Abcam).

Techniques: shRNA, Injection, Western Blot, Expressing, Immunostaining, Staining, Saline

Journal: Neurobiology of disease

Article Title: Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

doi: 10.1016/j.nbd.2022.105719

Figure Lengend Snippet: Fig. 1. Experimental design and BrainSpheres characterization. A) shows diagram of experimental procedure described in material and methods. B) shows confocal images of neuronal marker BTUBIII, dopaminergic neurons markers (KCNJ6, CALB1), astrocytes marker (GFAP) and oligodendrocyte marker (O4). Bars represent 50 μm.

Article Snippet: Subsequently, the BrainSpheres were washed twice with PBS (1×) and incubated with blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at 4 ◦C for 1 h. The BrainSpheres were washed with washing solution (1% BSA, 0.15% saponin in PBS), and incubated overnight at 4 ◦C with primary antibodies: anti- Calbindin 1 (CALB1) (1:200, for dopaminergic neurons) [ABclonal], anti-Potassium Inwardly Rectifying Channel Subfamily J Member 6 (KCNJ6) (1:200, for dopaminergic neurons)[ABclonal], anti-tyrosine hydroxylase (TH) (1:100, for dopaminergic neurons)[Millipore], anti-NF200 (1:200, neurofilament for all neurons) [Sigma] and anti-ZO-1 (1:200, endothelial tight junctions) [Life Technologies] diluted in blocking solution.

Techniques: Marker

Journal: Neurobiology of disease

Article Title: Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

doi: 10.1016/j.nbd.2022.105719

Figure Lengend Snippet: Fig. 4. Alteration in expression of dopaminergic neurons- and oxidative stress-related genes and extracellular levels of metabolites after 24 h treatment of Brain Spheres with 6-OHDA (500 μM), MPTP (5000 μM) and MPP+ (1000 μM). Gene expression of markers for dopaminergic neurons (TH, SLC6A3, CALB1, KCNJ6, TBR1), mitochondrial metabolism (ATP5O, NDUFB1, NDUFA1) and oxidative stress (GSTO1, KEAP1, SOD2) was analyzed by real-time qPCR. B) Extracellular metabolites measured by mass spectrometry (LC-MS/MS). C) Gene expression after compound washout and 5 days recovery period.In B and C bars represent mean ± SEM of 3–4 independent experiments. One-Way ANOVA followed by Kruskal–Wallis multiple comparison H tests was performed to evaluate statistical significance (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** P < 0.0001).

Article Snippet: Subsequently, the BrainSpheres were washed twice with PBS (1×) and incubated with blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at 4 ◦C for 1 h. The BrainSpheres were washed with washing solution (1% BSA, 0.15% saponin in PBS), and incubated overnight at 4 ◦C with primary antibodies: anti- Calbindin 1 (CALB1) (1:200, for dopaminergic neurons) [ABclonal], anti-Potassium Inwardly Rectifying Channel Subfamily J Member 6 (KCNJ6) (1:200, for dopaminergic neurons)[ABclonal], anti-tyrosine hydroxylase (TH) (1:100, for dopaminergic neurons)[Millipore], anti-NF200 (1:200, neurofilament for all neurons) [Sigma] and anti-ZO-1 (1:200, endothelial tight junctions) [Life Technologies] diluted in blocking solution.

Techniques: Expressing, Gene Expression, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: The Journal of biological chemistry

Article Title: Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts.

doi: 10.1016/j.jbc.2023.103049

Figure Lengend Snippet: Figure 2. Immunohistochemical staining of EPHX2 in tissues from Ephx2fx/fx and Ephx2-deficient mice. Expression of EPHX2 in heart, aorta, and kidney from Ephx2fx/fx Cre negative, Ephx2fx/fx/Tek-cre, Ephx2fx/fx/Myh6-cre, and global Ephx2−/−mice was detected by immunohistochemistry using a selective EPHX2 antibody (sc-22344). Data are representative of at least three mice per genotype group. EPHX, epoxide hydrolase.

Article Snippet: Membranes were probed with antibodies to EPHX2 (sc-25797, 1:1000) and ERK1 (sc-93, 1:2000) from Santa Cruz Biotechnology, β-actin (AC-74, 1:5000) and pERK1/ 2 (M8159, 1:1000) from Sigma, and phospho-S21/9-GSK3β (9331) and GSK3β (9315) (1:1000 each) from Cell Signaling.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Journal of translational medicine

Article Title: Central 5-HTergic hyperactivity induces myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)-like pathophysiology.

doi: 10.1186/s12967-023-04808-x

Figure Lengend Snippet: Fig. 4 Hypothalamic neuronal activity under 5-HT1Areceptor agonist treatment. After fluoxetine injection for 4 weeks, hypothalamic CRH neuronal activity (CRH, CRH_R2 and c-Fos) before and after challenge with 8-OH-DPAT was evaluated by immunofluorescent staining (A) and western blotting (B) analysis. In the hypothalamic plasma membrane, the protein expression levels of the 5-HT1A receptors, GIRK1 and GIRK2 were measured (C). The protein expression levels of BDNF, phosphorylated ERK1/2 and CREB in whole hypothalamic lysate were analyzed (D). The data are expressed as the mean ± SD (n = 4/group)

Article Snippet: To minimize nonspecific binding, the membranes were blocked in 5% bovine serum albumin for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies, including antibodies against the 5-HT1A receptor (1:200, ab85615, Abcam), TPH2 (1:500, NB100-74555, Novus), c-Fos (1:1000, ab208942, Abcam), CRH_R2 (1:200, NB10056485, Novus), CRH (1:500, 10944-1-AP, Proteintech), GIRK1 (1:100, MA5-25833, Invitrogen), GIRK2 (1:200, NB100-74575, Novus), p-ERK/ERK (1:200, #9101S/#9102S, Cell Signaling), p-CREB/CREB (1:200, ab32096/ab31387, Abcam), BDNF (1:200, ab108319, Abcam), vinculin (1:1000, sc-73614, Santa Cruz), α-tubulin (1:1000, ab7291, Abcam) and β-actin (1:1000, ab8229, Abcam).

Techniques: Activity Assay, Injection, Staining, Western Blot, Clinical Proteomics, Membrane, Expressing

Journal: Developmental biology

Article Title: Genetic Dissection of Midbrain Dopamine Neuron Development in vivo

doi: 10.1016/j.ydbio.2012.09.019

Figure Lengend Snippet: Quantification of cells in control and mutant mice reveals significant differences in the average number of TH+ and double positive neurons in the VTA and SNc. The average number of TH+ neurons and the 95% confidence intervals of counts from the VTA (A, B) and SNc (C, D) of control (dark grey shaded bars) and mutant mice (dark brown bars) in ventral, intermediate and dorsal planes. (A) The average number of TH+/Calb+ neurons in the VTA of mutant mice (light brown bars) was significantly lower than that of control mice (light grey bars) in all planes quantified. Calb+ neurons in the VTA indicated that the medial domain was not devoid of cells. (B) There were no double immunolabeled TH+/GIR2K+ neurons observed in the ventral VTA of any of the mutant GIRK2-stained slices consistent with the loss of VTA. (C) Although TH+ neurons were diminished in SNc, the average number of TH+/Calb+ neurons in the SNc was not significantly different between controls (light grey bars) and mutants (light brown bars) at any of the three horizontal planes. The blue arrowheads points to the average of TH+/Calb+ neurons. (D) The average number of TH+/GIRK2+ neurons in the SNc of mutant mice was significantly lower than that of control mice in all planes quantified. Images in A, C display TH+ (green), Calb+ (red) and TH+/Calb+ (yellow) cells while images in B, D display TH+ (green), GIRK2+ (red) and TH+/GIRK2+ (yellow) cells. Gray shading of bars indicates data from control animals with dark gray bars corresponding to average TH+ cell counts and nested light gray bars corresponding to average double positive cell counts. Brown shading indicates data from mutant animals with dark brown bars corresponding to average TH+ cell counts and nested lighter brown bars corresponding to average double positive cell counts.

Article Snippet: Calb indin (CALB) and g -protein i nward r ectifying potassium ( K ) channel 2 (GIRK2) were detected with anti-CALB (Swant; 1:1000) or anti-Girk2 (Alomone labs; 1:80), respectively.

Techniques: Mutagenesis, Immunolabeling, Staining

Journal: Journal of Parkinson's Disease

Article Title: Grafts Derived from an α -Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

doi: 10.3233/JPD-202366

Figure Lengend Snippet: Analysis of terminally differentiated DA neurons at day 35 in vitro . Immunostaining of VM-patterned neurons showing (A) high expression of neuronal markers TAU and MAP2, and DA marker TH. Immunostaining showing co-expression of (B) TH, FOXA2, and MAP2, (C) TH, GIRK2, and MAP2, and (D) TH, α Syn, and TAU. Scale bar 50 μ m. (E) Resting membrane potential (RMP) at day 35 measured by whole-cell patch-clamp recordings showing no major difference between the cell lines, n = 8 in each group. (F) Representative trace of voltage responses from the whole-cell patch-clamp showing evoked action potentials from current injection. (G) Inward sodium (Na + ) and outward potassium ( K + ) currents triggered by stepwise depolarization of the cell showing no major difference between the cell lines, n = 8 in each group. All data are expressed as mean±the standard deviation.

Article Snippet: The primary antibodies used were: rabbit anti-LMX1A (1:1000, Merck Millipore ab10533), mouse anti-FOXA2 (1:500, Santa Cruz Biotechnology sc101060), goat anti-OTX2 (1:2000, R&D Systems AF1979), rabbit anti-TH (1:1000, Merck Millipore ab152), mouse anti-TAU(HT7) (1:500, Thermo Fisher Scientific MN1000), chicken anti-MAP2 (1:10000, Abcam ab5392), goat anti-GIRK2 (1:200, Merck Millipore ab65096), and mouse anti- α -synuclein (1:250, BD Biosciences 610787).

Techniques: In Vitro, Immunostaining, Expressing, Marker, Patch Clamp, Injection, Standard Deviation