Journal: PloS one

Article Title: Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them.

doi: 10.1371/journal.pone.0006233

Figure Lengend Snippet: Figure 3. NSCs from prolonged culture in defined conditions can be efficiently differentiated into midbrain dopaminergic neurons. (A–E) Efficient differentiation into midbrain dopaminergic neurons by PA6-CM as shown by immunocytochemistry. The majority of the cells expressed b-III-tubulin and TH after 4 weeks of differentiation in PA6-CM (A–B). Co-expression of midbrain and A9 markers in TH+ dopaminergic neurons: Lmx1a (C), VMAT (D) and Girk2 (E). (F) Differential expression of dopaminergic markers in several stages of differentiation (NSC, dopaminergic precursors and dopaminergic neurons) by quantitative PCR. All the examined markers were up-regulated in dopaminergic populations compared to NSCs. doi:10.1371/journal.pone.0006233.g003

Article Snippet: The following primary antibodies were used: Oct4 (ab19857 AbCam) 1:1000; b-Tubulin isotype III clone SDL.3D10 (T8660 Sigma) 1:500; GalC (MAB342 Millipore) 1:50; GFAP (M0761 Chemicon) 1:50; Nestin (611658 BD Transduction laboratories) 1:500; Sox1 (AB5768 Chemicon) 1:200, TH (Pel-Freeze P40101) 1:500, TH clone TH-16 (T2928 Sigma) 1:1000; Lmx1a (a kind gift from Dr. Michael German, UCSF) 1:1500, Girk2 (AB5200 Chemicon) 1:250; Vmat2 (PPS089 R&D) 1:1000, GABA (ab8891 Abcam) 1:500, L-Glutamate (ab8889 Abcam) 1:500, DBH (AB1585 Chemicon) 1:1000, HB9 (81.5C10 DSHB) 1:50, and as secondary antibodies: Alexa Fluor 488 Goat Anti-Mouse, Alexa Fluor 594 Goat Anti-Mouse, Alexa Fluor 488 Goat AntiRabbit, Alexa Fluor 594 Goat Anti-Rabbit.

Techniques: Immunocytochemistry, Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction

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: 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 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: Hypothalamic neuronal activity under 5-HT 1A receptor 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-HT 1A 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-HT 1A receptor (1:200, ab85615, Abcam), TPH2 (1:500, NB100-74555, Novus), c-Fos (1:1000, ab208942, Abcam), CRH_R2 (1:200, NB100-56485, 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, Membrane, Expressing

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 2. GIRK1 and GIRK2 protein expression levels shown in untransfected HEK293 cells or cells engineered to express GIRK2, GIRK1/2, or GIRK2 and NPY4R. The full gel is shown in Supporting Information Figure S1.

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 3. Characterization of VU0529331 efficacy and potency using Tl+ flux. VU0529331 activated GIRK2 and GIRK1/2 channels in a concentration-dependent manner. In contrast, VU0466551 only activated GIRK1-containing channels. Potency (EC50) values were provided for active compounds. Both compounds were inactive in untransfected HEK293 cells. GIRK2 channel activity was normalized to a maximally effective concentration of VU0529331, while GIRK1/2 channel activity was normalized to a maximally effective concentration of VU0466551. All data shown are representative of, at minimum, three independent experiments. Error bars represent the standard error of the mean (SEM).

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Concentration Assay, Activity Assay

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 4. (a) Pertussis toxin (PTX) does not affect the activity of VU0529331 on G2Y4 cells while (b) activation of GIRK2 through the Gβγ-subunit after GPCR activation is fully inhibited. The G2Y4 cells express neuropeptide Y receptor type 4 (NPY4R) in addition to any GPCRs native to HEK293 cells. NPY4R was activated using a maximally effective concentration, 200 nM, of human pancreatic polypeptide (hPP). All values in (a) were normalized to the predicted maximum activity of VU0529331 under VHL conditions. VU0529331 activity in (b) is normalized data from the 30 μM value in (a). 95% confidence intervals for the EC50 measurements were calculated using a 2-tailed Mann−Whitney test. **** indicates p < 0.0001. Error bars represent the standard error of the mean (SEM).

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Activity Assay, Activation Assay, Concentration Assay, MANN-WHITNEY

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 6. VU0529331 at 80 μM had no effect on the rectification ratio of HEK293 cells expressing (a) GIRK2 and (b) GIRK1/2 channels. Whole-cell patch-clamp electrophysiology recordings were generated under 20 mM K+ external buffer solution containing 0.25% (v/v) DMSO (VHL). A Wilcoxon matched-pairs signed rank test analysis indicated that there was not a statistically significant difference between the two conditions in (a) or (b).

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Patch Clamp, Generated

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 5. VU0529331 at 80 μM significantly increased both inward and outward currents in HEK293 cells expressing (a) GIRK2 and (b) GIRK1/2 channels. VU0529331 did not evoke currents in (c) untransfected HEK293 cells. (d) The increase in current density for GIRK1/2 and GIRK2 cells at −10 mV was significantly different. For each cell type in (a)−(c), the conditions were compared using a two-way ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The fold differences in (d) were compared using a Welch’s t test. Recordings were generated in 20 mM K+ external buffer solution containing 0.25% (v/v) DMSO (VHL). Error bars indicate standard error around the mean (SEM).

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Generated

Journal: ACS chemical neuroscience

Article Title: Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

doi: 10.1021/acschemneuro.8b00287

Figure Lengend Snippet: Figure 7. Whole-cell patch-clamp electrophysiology using either untransfected HEK293 cells or cells engineered to express GIRK channels. Currents were held at −60 mV while cells were bathed in 20 mM K+ external buffer. Exposure of (a) GIRK2 cells or (d) GIRK1/2 cells to escalating VU0529331 (VU331) concentrations showed concentration-dependent increases in inward current density. (b) Untransfected HEK293 cells did not show any changes in currents after VU331 exposure. Both (c) GIRK2 and (e) GIRK1/2 currents evoked by maximally active VU331 concentrations were blocked by exposure to extracellular solutions containing 2 mM barium (Ba2+), the nonselective inwardly rectifying K+ channel inhibitor.

Article Snippet: Next, the membrane was incubated overnight at 4 °C in TT20 containing a 1:2000 dilution of a monoclonal IgG2b mouse anti-GIRK1 antibody (cat#TA504152, Origene, Rockville, MD) and a 1:1000 dilution of monoclonal IgG rabbit anti-GIRK2 antibody (cat#25797, Cell Signaling Technology, Danvers, MA).

Techniques: Patch Clamp, Concentration Assay

Journal: Life

Article Title: Pharmacological Characterization of Veldoreotide as a Somatostatin Receptor 4 Agonist

doi: 10.3390/life11101075

Figure Lengend Snippet: Analysis of exogenous ( A ) SST2, ( B ) SST4, or ( C ) SST5 receptors co-expressed with GIRK2 channels in HEK293 cells. Cells were exposed to 10 −12 M to 10 −6 M SS-14 or veldoreotide. Concentration-response curves were generated using data from three to four independent experiments performed in duplicate (mean ± SEM). Changes in fluorescence with agonists were subtracted from changes in fluorescence with vehicle. GIRK = G-protein-coupled inwardly rectifying potassium; RFU = relative fluorescence units; SEM = standard error of the mean; SST = somatostatin.

Article Snippet: A custom-made plasmid pCMV vector encoding the human G-protein-coupled inwardly rectifying potassium 2 (GIRK2) channel along with a green fluorescence protein (GFP) tag were obtained from OriGene (Rockville, MD, USA), as previously described [ ].

Techniques: Concentration Assay, Generated, Fluorescence

Journal: Life

Article Title: Pharmacological Characterization of Veldoreotide as a Somatostatin Receptor 4 Agonist

doi: 10.3390/life11101075

Figure Lengend Snippet: Agonist-selective G-protein signaling in HEK293 cells.

Article Snippet: A custom-made plasmid pCMV vector encoding the human G-protein-coupled inwardly rectifying potassium 2 (GIRK2) channel along with a green fluorescence protein (GFP) tag were obtained from OriGene (Rockville, MD, USA), as previously described [ ].

Techniques: