Journal: Molecular Pain

Article Title: Peripheral nerve injury induces loss of nociceptive neuron-specific Gαi-interacting protein in neuropathic pain rat

doi: 10.1177/1744806916646380

Figure Lengend Snippet: Primary antibodies and IB4 used for IHC in this study.

Article Snippet: GABA B receptor 2 , Rabbit polyclonal , Alomone Labs/AGB002 , 1:50.

Techniques:

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a , b Heat maps revealing differential gene expression in FMT-DSS vs FMT-CON, FMT-DSS + SP vs FMT-DSS groups. n = 4 mice/group. The pathway-related genes were selected (log2 fold change at least > 1, p < 0.05). c , d KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS group compared with the FMT-CON group (Top 20). n = 4 mice/group. e , f KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS + SP group vs the FMT-DSS group (Top 20). n = 4 mice/group. g Quantitative real-time PCR analysis of mRNA expressions of NF-κB signaling pathway genes ( p65 and IKBα ) in the hippocampus. n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). h The protein expression of p-p65 and p-IKBα in hippocampus tissue, as determined by western blotting. n = 3 independent experiments. i Quantitative real-t i me PCR analysis of mRNA expressions of GABA receptor and Ca 2+ signaling genes ( Gabra1 , Gabra3 , Gabrg2 , Gabrb2 and Camk2d ). n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). j Protein expressions of GABA A R, GABA B R, GAD65 and CaMKII were measured in hippocampus tissue by western blot. n = 3 independent experiments. k Representative immunofluorescence images of double-labeling for p-IKBα (red)/Iba1 (green) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. I Representative immunofluorescence images of double-labeling for GABA A R (green)/GFAP (red) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. m Double immunofluorescence staining for CaMKII (green)/GFAP (red) in hippocampus tissues and statistical analysis. Scale bar = 50 μm. n = 3 independent experiments. n Differential gene expressions were presented by the heatmap in microglia of three groups, and GSEA analysis of microglia from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. o Heat maps of differential gene expression in astrocytes from three groups and GSEA analysis of astrocytes from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Labeling, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Diagram illustrating the mouse model of colitis employed in this study, and inositol treatments are indicated (Some schematic elements were created by Figdraw.com). b Daily body weight changes. n = 8 mice/group. c Diseases activity index. n = 6 mice/group. d Macroscopic pictures of colons. e H&E-stained colon sections. Scale bar = 100 μm. n = 5 independent experiments. f Immunofluorescence staining for E-cadherin (red)/DAPI (blue) in colon tissues. Scale bar = 100 μm. n = 4 independent experiments. g The mRNA level of inflammatory cytokines ( IL-1β , IL-6 and TNF-α ) in the colon. n = 4 mice/group. h , i Representative movement tracks in the open field test and related bar graphs (Distance traveled, distance of center region and speed). n = 6 mice/group. j , k Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms and percentage of times entering the open arms. n = 6 mice/group. l Representative H&E-stained hippocampus sections of three groups. Scale bar = 100 μm. n = 6 independent experiments. m Photomicrographs of Nissl staining in the hippocampus. Scale bar = 100 μm. n = 6 independent experiments. n The mRNA expressions of downstream cytokines ( IL-6 , IL-1β , TNF-α and IL-10 ) in the hippocampus tissue. n = 5 mice/group. o Representative immunohistochemistry images of Iba-1 in hippocampus, Scale bar = 100 μm. n = 3 independent experiments. p The concentration of GABA in hippocampus tissue. n = 6 mice/group. q The mRNA expression of GABA A Rα1 in hippocampus. n = 5 mice/group. r The protein level of GABA B R detected by western blot. n = 3 independent experiments. s Double immunofluorescence staining for GFAP (red)/GAT1 (green) in the DG and CA1 regions of hippocampus tissue. Scale bar = 50 μm. n = 3 independent experiments. Data were presented as means ± SD. For body weight change, two-way repeated-measures ANOVA was performed and the rest of the statistics was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Activity Assay, Staining, Immunofluorescence, Immunohistochemistry, Concentration Assay, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Schematic illustration of experimental design (Some schematic elements were created by Figdraw.com). b , c Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms, percentage of distance traveled in the open arms and percentage of times entering the open arms. n = 6 mice/group. d , e Track plot of the elevated zero maze, and statistical analysis including percentage of time spent in the open zones and percentage of times entering the open zones. n = 6 mice/group. f Statistical analysis, including percentage of distance traveled in the light area, percentage of time spent in the light area, and times of transitions in the light-dark box test. n = 6 mice/group. g Representative images of H&E-staining for the hippocampus sections. Scale bar = 100 μm. n = 6 independent experiments. h Representative images of Nissl staining for the hippocampus sections. Scale bar = 100 μm. n = 4 independent experiments. i The content of inositol in hippocampus tissue tested by ELISA. n = 8 mice/group. j The content of inositol in serum tested by ELISA. n = 8 mice/group. k–n The mRNA expression of TNF-α , IL-1β , IL-6 and iNOS in BV-2 cells. n = 3 mice/group. o, p The mRNA expression of Gabra3 and Camk2d in C8D1A cells. n = 3 mice/group. q The protein level of GABA A R, CaMKII, GABA B R detected by western blot. n = 3 independent experiments. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a The diagram of the calyx of Held synapse. b Strategy for selective deletions of all neurexins at the calyx of Held by crossing PV-Cre mice with triple Nrxn123 cKO mice (Chen et al. , Luo et al. ). c Representative traces of EPSC before and after application of 20 µM SKF-97541 (SKF), a potent and selective GABA B -receptor agonist, recorded in acute slices from littermate control and neurexin123 TKO mice at P12–P14. The normalized EPSCs before and after SKF are shown in inset. d Summary graphs of EPSC1 amplitudes before and after SKF for control and Nrxn123 TKO mice. P = 2.3E−7, P = 0.072, paired two-sided t -test. P = 0.0062, unpaired two-sided t -test. e Summary graphs of EPSC1 remaining unblocked by SKF application. P = 1.79E−7, unpaired two-sided t -test. f Summary graphs of the paired-pulse ratio (PPR) before and after SKF in control and Nrxn123 TKO mice. P = 2.81E−5, P = 0.09, paired two-sided t -test. P = 0.0038, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( d – f ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative confocal microscopy images of MNTB-containing brainstem slice with specific labeling of VGluT1 (green) and GABA B -receptor subunit 2 (GABA B2 , red) from both littermate control and Nrxn123 TKO mice at P12. Scale bar, 10 µm. b Summary of VGluT1 and GABA B2 immunostaining intensity (normalized to control). P = 1, P = 1.35E−9, unpaired two-sided t -test. c , d Same as a and b except for specific labeling of VGluT1 (green) and GABA B -receptor subunit 1 (GABA B1 , red). P = 1, P = 0.031, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , d ). Statistical differences were assessed by Student’s t -test. (* P < 0.05; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Confocal Microscopy, Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Representative dSTORM images of MNTB-containing brainstem slice with dual labeling of Homer1 (magenta) and GABA B -receptor subunit 2 (GABA B2 , yellow) from both littermate control and Nrxn123 TKO mice. b Summary of the number of GABA B2 clusters normalized to the number of Homer1 clusters. P = 0.0051, unpaired two-sided t -test. c Distribution of Homer1 and GABA B2 localizations. d – f Similar as a – c , except for GABA B1 antibody used. P = 0.0057, unpaired two-sided t -test. Data are means ± SEM. Number of sections/animals for immunostaining are indicated in the bars ( b , e ). Statistical differences were assessed by Student’s t -test. (** P < 0.01). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Labeling, Control, Immunostaining

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental approach for virus injection and electro-physiology recording (Top); GFP expression in AAV-transfected CA3 region of hippocampus (bottom). b Representative traces of EPSCs evoked by fiber stimulations with increased intensity, recorded in acute hippocampus slices from Nrxn123 cKO mice injected with AAV-ΔCre or AAV-Cre. c The input–output curve of EPSC amplitudes in relation to the stimulation intensity. P = 0.0113, unpaired two-sided t -test. d Example traces of paired-pulse EPSCs before and after addition of GABA B -receptor agonist SKF, the normalized EPSCs before and after SKF are shown in inset. The intensity of fiber stimulation was tuned to evoke EPSC1 at similar amplitude for each cell. e Summary graphs of EPSC1 amplitudes before and after SKF for control (ΔCre) and Nrxn123 TKO synapses (Cre). P = 0.0001, P = 0.0001, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. f Summary graphs of EPSC1 rise time (left) and decay time constants (right). P = 0.0008, P = 0.6136, unpaired two-sided t -test. g Summary graphs of EPSC1 remaining unblocked by SKF application. P = 0.0013, unpaired two-sided t -test. h Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0984, paired two-sided t -test. P = 0.2772, unpaired two-sided t -test. i Summary graphs of the CV of EPSC1 amplitude before and after SKF in control and Nrxn123 TKO synapses. P = 0.0385, P = 0.99, paired two-sided t -test. P = 0.4664, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the graph ( c ) or bars ( e – i ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01, *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of experimental setup for virus injection and optogenetic stimulation of PV + interneurons (Top); PV-Cre dependent expression of Chief-TdTomato in AAV-transfected CA1 region of hippocampus (bottom). b Example traces of paired-pulse blue light-evoked IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute hippocampus slices from PVCre + mice (control) or PVCre + /Nrxn123 cKO mice (TKO) injected with AAV-DIO-Chief-TdTomato. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0001, P = 0.0026, paired two-sided t -test. P = 0.6, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF. P = 0.0047, unpaired two-sided t -test. e Summary graphs of the PPR ratio before and after SKF in control and Nrxn123 TKO synapses. P = 0.0022, P = 0.6165, paired two-sided t -test. P = 0.5114, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude. P = 0.0143, P = 0.99, paired two-sided t -test. P = 0.99, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.4974, P = 0.063, unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Virus, Injection, Expressing, Transfection, Control

Journal: Nature Communications

Article Title: Neurexins regulate presynaptic GABA B -receptors at central synapses

doi: 10.1038/s41467-021-22753-5

Figure Lengend Snippet: a Schematic of cerebellum circuits and experimental approach for stimulating basket cell (BC)-Purkinje cell (PC) inhibitory synapse. b Example traces of paired-pulse IPSCs before and after addition of GABA B -receptor agonist SKF, recorded in acute cerebellar slices from littermate control and Nrxn123 TKO mice P35-49. The normalized IPSCs before and after SKF are shown in inset. c Summary graphs of IPSC1 amplitudes before and after SKF for control and Nrxn123 TKO synapses. P = 0.0016, P = 0.051, paired two-sided t -test. P = 0.0389, unpaired two-sided t -test. d Summary graphs of IPSC1 amplitude remaining unblocked by SKF for control and Nrxn123 TKO mice. P = 0.0028; unpaired two-sided t -test. e Summary graphs of the PPR before and after SKF in control and Nrxn123 TKO mice. P = 0.000101, P = 0.097, paired two-sided t -test. P = 0.762, unpaired two-sided t -test. f Summary graphs of the CV of IPSC1 amplitude before and after SKF in control and Nrxn123 TKO mice. P = 0.00073, P = 0.066, paired two-sided t -test. P = 0.348, unpaired two-sided t -test. g Summary graphs of IPSC1 rise time (left) and decay time constant (right). P = 0.000953, P = 0.977; unpaired two-sided t -test. Data are means ± SEM. Number of cells (from at least three mice per group) analyzed are indicated in the bars ( c – g ); Statistical differences were assessed by Student’s t -test. (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against VGluT1 (guinea pig, polyclonal, 1:1000, Millipore, Cat#: AB5905; RRID: AB_2301751), GABA B1 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-183), and GABA B2 (mouse, monoclonal, 1:500, NeuroMab Cat#: 75-125) were used.

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Neuronal Chloride Regulation via KCC2 Is Modulated through a GABA B Receptor Protein Complex

doi: 10.1523/JNEUROSCI.2164-16.2017

Figure Lengend Snippet: GABA B Rs associate with KCC2 at the cell membrane in cortex and hippocampus. A , Affinity purification and mass spectrometry were used to identify proteins associated with the GABA B R in synaptic membrane preparations from adult rat cortex. The table shows the number of unique peptides for each protein, obtained across three independent isolates of the GABA B R1 protein. Where the identified proteins have previously been shown to associate with the GABA B R, references are provided. B , KCC2 coimmunoprecipitates with the GABA B R. Rat hippocampal lysates were immunoprecipitated with an anti-GABA B R1 antibody, and subsequent Western blot analysis for KCC2 revealed two distinct bands at ∼130 and 270 kDa, which correspond to the monomeric and dimeric forms of KCC2 (top blot, right lane). Probing for GABA B R1 confirmed successful immunoprecipitation of both the GABA B R1a and GABA B R1b isoforms (bottom blot). In contrast, controls using sheep anti-IgG antibody failed to pull down KCC2 or GABA B R1 (middle lanes). C , The GABA B R coimmunoprecipitates with KCC2. Immunoprecipitates from rat hippocampal lysates isolated with an anti-KCC2 antibody were positive for GABA B R1a, GABA B R1b, and KCC2 (top and bottom blots, right lanes). In contrast, control experiments using a rabbit anti-IgG failed to pull down KCC2 or GABA B R1 from the same lysate (middle lanes). D , The GABA B R (left) and KCC2 (right) are both localized at the plasma membrane of pyramidal neurons, as revealed by rabbit polyclonal antibodies against GABA B R2 (left) and KCC2 (right) in separate rat organotypic hippocampal slices (P7 + 7–14 DIV). Scale bars, 10 μm. E , Using a sequential double-labeling technique in dissociated neuronal cultures (see Materials and Methods), GABA B R2 (red) and KCC2 (green) were found to be colocalized (yellow) at somatic and dendritic membranes of hippocampal pyramidal neurons. Magnifications of the areas highlighted within the white boxes are provided in the panels below. Control staining (bottom) for GABA B R2 and β-tubulin revealed nonoverlapping signals. Scale bars, 20 and 5 μm.

Article Snippet: The following antibodies were used in this study: sheep anti-GABA B R1 antibody , mouse anti-GABA B R1 (University of California–Davis/National Institutes of Health NeuroMab Facility, 75-183, clone N93A/49; Sigma, WH0002550M1-100UG, clone 2D7), rabbit anti-GABA B R2 (GlaxoSmithKline) , sheep IgG (Thermo Fisher Scientific, 31243), rabbit anti-C terminus KCC2 (Millipore, 07-432), rabbit anti-β-tubulin (Sigma, T2200) rabbit anti-GFP (Thermo Fisher Scientific, A11122), rabbit IgG (Thermo Fisher Scientific, 31235), mouse anti-β-tubulin (Cambridge Bioscience, MMS-435P-250), mouse anti-transferrin receptor (Thermo Fisher Scientific, 13-6890), mouse anti-NKCC1 (Developmental Studies Hybridoma Bank, T4 clone), mouse anti-actin (Sigma, A3854), HRP-conjugated donkey anti-rabbit (Stratech, 711-055-152), HRP-conjugated goat anti-mouse (Stratech, 115-055-166), donkey anti-rabbit Cy3-conjugated monovalent Fab fragment (Stratech, 111-167-003), and donkey anti-rabbit Alexa-488-conjugated secondary (Thermo Fisher Scientific, A21206).

Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation, Western Blot, Isolation, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Neuronal Chloride Regulation via KCC2 Is Modulated through a GABA B Receptor Protein Complex

doi: 10.1523/JNEUROSCI.2164-16.2017

Figure Lengend Snippet: The GABA B R associates with the transmembrane domain of KCC2. A , The GABA B R1 monoclonal antibody 2D7 used for immunoblotting is specific for GABA B R1. In Western blots of total rat brain and rat hippocampus homogenate, two bands corresponding to GABA B R1a and GABA B R1b were detected. Blots of lysates from CHO cells stably expressing either GABA B R1a/R2 or GABA B R1b/R2 revealed multiple bands, consistent with differential glycosylation of GABA B R1 proteins in this system. B , The KCC2-GABA B R association can be reconstituted in a heterologous cell system. CHO cells stably expressing GABA B R1b/R2 were transiently transfected with FL-KCC2-GFP and used for coimmunoprecipitation experiments. Western blots of resulting complexes showed that KCC2 can be coimmunoprecipitated with GABA B R1b (left), and the reciprocal coimmunoprecipitation confirms the association (right). IgG controls were included in each experiment. Sh, Sheep; Rab, rabbit. C , Schematic diagram of KCC2 showing the intracellular NTD, CTD, and TMD with its 12 predicted transmembrane helices. D , GFP fusion proteins of FL-KCC2 and different KCC2 deletion constructs containing the TMD are expressed and transported to the plasma membrane. Biotinylation experiment comparing total (T) and cell surface (S) protein levels in CHO GABA B R1b/R2 cells, transiently transfected with different GFP fusion constructs (left). FL-KCC2 (predicted molecular mass 150 kDa, although the TM region is glycosylated), TMD + CTD (predicted 140 kDa), NTD + TMD (predicted 96 kDa), and TMD (predicted 86 kDa) are all detected on the cell surface, whereas GFP (27 kDa) alone is not. Additional bands detected likely represent alternatively glycosylated, degraded, or aggregated proteins. The blot was reprobed with GABA B R1 antibody to confirm surface expression of the receptor (bottom). E , The transmembrane domain of KCC2 is required for the association with the GABA B R. Coimmunoprecipitation experiments on CHO GABA B R1b/R2 cells transiently expressing KCC2-GFP deletion constructs were performed using anti-GFP as the precipitating antibody. When the resulting complexes were probed for GABA B R1 (top), all KCC2-GFP fusion proteins containing the TMD successfully coimmunoprecipitated GABA B R1b. However, GFP fusion proteins containing only the NTD (predicted 38 kDa) or the CTD (predicted 82 kDa) did not capture GABA B R1b. Under these conditions, and compared with FL-KCC2 (100%), the relative amounts of GABA B R detected after isolation with the GFP antibody was 68 ± 16% for NTD + TMD, 36 ± 2% for TMD + CTD, 76 ± 19% for TMD, 3 ± 1% for NTD, 3 ± 2% for CTD, and 3 ± 1% for GFP ( n = 2–4 in each case). These quantifications will be influenced by the level of expression of each of the different constructs. Cell lysates expressing the GFP fusion proteins are shown (bottom). Additional bands detected with the GFP antibody are likely to be alternatively glycosylated, degraded, or higher-order aggregates of the expressed fusion proteins. F , Control experiments show that the endogenous 100 kDa transferrin receptor (TfR) is not coimmunoprecipitated with KCC2-GFP proteins in CHO GABA B R cells (top; IgG bands are shown for clarity). Lysates are also shown (bottom).

Article Snippet: The following antibodies were used in this study: sheep anti-GABA B R1 antibody , mouse anti-GABA B R1 (University of California–Davis/National Institutes of Health NeuroMab Facility, 75-183, clone N93A/49; Sigma, WH0002550M1-100UG, clone 2D7), rabbit anti-GABA B R2 (GlaxoSmithKline) , sheep IgG (Thermo Fisher Scientific, 31243), rabbit anti-C terminus KCC2 (Millipore, 07-432), rabbit anti-β-tubulin (Sigma, T2200) rabbit anti-GFP (Thermo Fisher Scientific, A11122), rabbit IgG (Thermo Fisher Scientific, 31235), mouse anti-β-tubulin (Cambridge Bioscience, MMS-435P-250), mouse anti-transferrin receptor (Thermo Fisher Scientific, 13-6890), mouse anti-NKCC1 (Developmental Studies Hybridoma Bank, T4 clone), mouse anti-actin (Sigma, A3854), HRP-conjugated donkey anti-rabbit (Stratech, 711-055-152), HRP-conjugated goat anti-mouse (Stratech, 115-055-166), donkey anti-rabbit Cy3-conjugated monovalent Fab fragment (Stratech, 111-167-003), and donkey anti-rabbit Alexa-488-conjugated secondary (Thermo Fisher Scientific, A21206).

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Isolation

Journal: Journal of Neuroinflammation

Article Title: Mesenchymal-derived extracellular vesicles enhance microglia-mediated synapse remodeling after cortical injury in aging Rhesus monkeys

doi: 10.1186/s12974-023-02880-0

Figure Lengend Snippet: The expression of inhibitory synaptic markers in perilesional M1 and PMC. a Box-and-whisker plots with vertical scatter plots of individual cases showing the particle density and average size of VGAT+ puncta in perilesional M1 and in PMC (Ctr: n = 3; Veh group: n = 5; EV group: n = 5 monkeys). b Representative maximum-projection confocal images of VGAT immuno-label in M1 and PMC. c Box-and-whisker plots with vertical scatter plots of individual cases of the density and average size of GABA a α1+ puncta in perilesional M1 and in PMC. The density of GABA a α1 subunit in perilesional M1 was significantly lower in both veh and EV group as compared with non-lesion controls (one-way ANOVA, main effect, p = 0.004; Fisher’s LSD post hoc, ctr. vs. Veh, p = 0.038; ctr. vs. EV, p = 0.009). The density of GABA a α1 in PMC was lower in veh and EV group (one-way ANOVA, main effect, p = 0.016; Fisher’s LSD post hoc, ctr. vs. veh, p = 0.05; ctr. vs. EV, p = 0.02). The size of GABA a α1 in M1 was significantly smaller in the EV group ( p < 0.001). d Representative maximum-projection confocal images of GABA a α1 receptor subunit immuno-label in M1 and PMC. e Box-and-whisker plots with vertical scatter plots of individual cases showing the particle density and average size of GABA b R2+ puncta in perilesional M1 and PMC. f Representative maximum-projection confocal images of GABA b R2 subunit immuno-label in M1 and PMC. For c-f Ctr: n = 3; Veh group: n = 4; EV group: n = 5 monkeys. Scale bar: 20 µm. Box-and-whisker plots: bars show interquartile range and median (horizontal line) with error bars = 95% confidence interval; * p < 0.05, ** p < 0.01

Article Snippet: Sections were incubated in primary antibodies in carrier solution (0.2% BSA, 1% NDS, 0.1% Triton X-100 in 0.1 M PB) at 4 °C for 72 h. We used primary antibodies to goat VGLUT1 (1: 500, Synaptic Systems, Cat# 135,307, RRID: AB_2619821), guinea pig VGLUT2 (1:1000, Synaptic Systems, Cat# 135,404, RRID: AB_887884), rabbit GLUR2/3 (1:500, Millipore Cat# 07–598, RRID:AB_11213931), guinea pig VGAT (1:400, Synaptic Systems, Cat# 131,004, RRID: AB_887873), rabbit GABA a α1 (1:500, Abcam, AB33299, RRID: AB_732498), mouse GABA b R2 (1:1000, LSBio, LS-C285897).

Techniques: Expressing, Whisker Assay

Journal: Journal of Neuroinflammation

Article Title: Mesenchymal-derived extracellular vesicles enhance microglia-mediated synapse remodeling after cortical injury in aging Rhesus monkeys

doi: 10.1186/s12974-023-02880-0

Figure Lengend Snippet: Glutamate and GABA receptors subunit mRNA expression in perilesional cortex. a Box-and-whisker plots with vertical scatter plots of individual cases showing relative fold changes in glutamate receptor subunit gene expression. The gene expression of GRIA2 (GLUR2) was significantly higher in the veh group, as compared with non-lesion controls ( t -test, p < 0.001) and the EV group ( p = 0.01). b Fold changes of GABA receptor subunit gene expression. The gene expression of GABRD (GABA a ∂) was significantly higher in the EV group as compared with non-lesion controls (t -test, p = 0.004). Gene names: GRIA1 (AMPA GLUR1), GRIA2 (AMPA GLUR2), GRIN1 (NMDA NR1), GRIN2B (NMDA NR2B), GABRA1 (GABAa α1), GABRA2 (GABA a α2), GABRA5 (GABA a α5), GABRD (GABA a ∂), GABBR2 (GABA b R2). Ctr: n = 2; Veh: n = 5; EV: n = 4 monkeys. c MDS plot showing clustering of cases based on mRNA expression profiles of Glu and GABA receptor subunits. The proximity of points indicates the relative similarity-based pair-wise correlation of these multiple mRNA expression variables. Ctr: n = 2; Veh: n = 5; EV: n = 3 monkeys. Box-and-whisker plots: bars show interquartile range and median (horizontal line) with error bars = 95% confidence interval; * p < 0.05, ** p < 0.01

Article Snippet: Sections were incubated in primary antibodies in carrier solution (0.2% BSA, 1% NDS, 0.1% Triton X-100 in 0.1 M PB) at 4 °C for 72 h. We used primary antibodies to goat VGLUT1 (1: 500, Synaptic Systems, Cat# 135,307, RRID: AB_2619821), guinea pig VGLUT2 (1:1000, Synaptic Systems, Cat# 135,404, RRID: AB_887884), rabbit GLUR2/3 (1:500, Millipore Cat# 07–598, RRID:AB_11213931), guinea pig VGAT (1:400, Synaptic Systems, Cat# 131,004, RRID: AB_887873), rabbit GABA a α1 (1:500, Abcam, AB33299, RRID: AB_732498), mouse GABA b R2 (1:1000, LSBio, LS-C285897).

Techniques: Expressing, Whisker Assay