Anti-GABA B Receptor 1 抗体 [2D7]

• WB

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Western blot - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

GABA B Receptor 1 antibody (ab55051) at 1ug/lane + IMR-32 cell lysate at 25ug/lane.

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All lanes:

Western blot - Anti-GABA B Receptor 1 antibody [2D7] (ab55051)

Predicted band size: 108 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

ICC/IF image of ab55051 stained SHSY5Y cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab55051, 5μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

This image was generated using the ascites version of the product.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

ab55051 staining GABA B receptor 1 in SK-N-SH cells treated with NMDA (ab120052), by ICC/IF. Internalization of GABA B receptor 1 correlates with increased concentration of NMDA, as described in literature.
The cells were incubated at 37°C for 30 minutes in media containing different concentrations of ab120052 (NMDA) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab55051 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

This image was generated using the ascites version of the product.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

ab55051 staining GABA B receptor 1 in SK-N-SH cells treated with L-Glutamate (ab120049), by ICC/IF. Internalization of GABA B receptor 1 correlates with increased concentration of L-Glutamate, as described in literature.
The cells were incubated at 37°C for 30 minutes in media containing different concentrations of ab120049 (L-Glutamate) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab55051 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

This image was generated using the ascites version of the product.

• Flow Cyt

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Flow Cytometry - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

Overlay histogram showing SH-SY5Y cells stained with ab55051 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab55051, 0.5μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This image was generated using the ascites version of the product.

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

Immunocytochemistry-immunofluorescence using Anti-GABA B Receptor 1 antibody [2D7], ab55051. Publication image from Niere, F. et al., 2016, Nat Commun, 27666021. Legend direct from paper.

Acute ethanol increases dendritic GABABRs in hippocampus.(a–c) Western blot analyses of GABABR1 and GABABR2 in isolated hippocampal synaptoneurosomes from ethanol-treated (ETOH; 2.5 g kg−1 i.p.), and vehicle-treated (Veh; saline i.p.) C57BL/6 male mice 30 min post injection. (a) Pseudocoloured representative western blots to show intensity with normalized optical density for each band indicated below blot (Lookup table, below western blot). No significant change was observed in b GABABR1, but a significant increase was found in c GABABR2 with ethanol treatment. Western blots were normalized to the loading control,α-Tubulin. GABABR1 : Veh=1.00±0.11; ETOH=1.19±0.08. Experiment was repeated five times. GABABR2 : Veh=1.00±0.07; ETOH=1.37±0.05. (d,e) Representative immunostaining images of GABABR1 and GABABR2 in cultured rat hippocampal dendrites normalized to microtubule associated protein 2 (MAP2) as volume control. There was no change in f GABABR1 and a significant increase in g GABABR2 in ethanol treated (30 mM, 2 h) compared with vehicle-treated (H2O, 2 h) dendrites : Total GABABR1 : Veh=1.00±0.03, n=46 dendrites; ETOH=1.04±0.04, n=51 dendrites. Total GABABR2 : Veh= 1.00±0.03, n=46 dendrites; ETOH=1.47±0.05, n=51 dendrites. (h,i) Immunofluorescence shows a significant increase in surface GABABR1 expression in dendrites of cultured rat hippocampal neurons treated with ethanol (30 mM, 2 h); (i) Surface expression of GABABR1 in vehicle-treated (H2O, 2 h) and ethanol-treated (30 mM, 2 h) dendrites. Veh=1.00±0.09, n=43 dendrites; ETOH=1.66±0.12, n=47 dendrites. Significance determined by Student's t-test. Values represent mean±s.e.m. Scale bars, 5 µm. Uncropped version of western blots, with size markers are available in Supplementary Fig. 7a.

• WB

CiteAb

Western blot - Anti-GABA B Receptor 1 antibody [2D7] (AB55051)

Western Blotting using Anti-GABA B Receptor 1 antibody [2D7], ab55051. Publication image from Niere, F. et al., 2016, Nat Commun, 27666021. Legend direct from paper.

GABABR1 and GABABR2 mRNAs are FMRP targets.(a,b) RNA immunoprecipitation (RIP) for FMRP was performed using brains from wild type (WT) and Fmr1 KO male mice. (a) Gels showing RT–qPCR amplified product of input sample, FMRP RIP, and IgG control for GABABR1 and GABABR2. (b) Relative fold-enrichment as determined by real-time qPCR relative to input control (δδCt=2−(Ct FMRP RIP−Ct IgG RIP)−(Ct FMRP input−Ct IgG input)). FMRP binds GABABR1, GABABR2, and the positive control CaMKIIα mRNA as detected in the RIP sample by real-time qPCR. Cacnα2δ2 served as a negative control and was not detected above background. WT : GABABR1=2.66±0.248, n=2; GABABR2=2.19±0.08, n=2; CaMKII=3.72±0.94, n=2; Cacnα2δ2=0.11±0.6, n=2. Fmr1 KO : GABABR1=0.01±0.0002, n=2; GABABR2=0.02±0.00006, n=2; CaMKII±0.04±0.01, n=2; Cacnα2δ2=0.012±0.005, n=2. (c–g) Western blot analysis of hippocampal synaptoneurosomes isolated from C57BL/6 WT and Fmr1 KO mice on a C57BL/6 background indicates the absence of (e) FMRP and increased protein expression of (f) GABABR1 and (g) GABABR2. Representative western blots are pseudocoloured to indicate intensity of bands, and the normalized optical density for each band is indicated below blot (Lookup table, below western blot). Western blots were normalized to the loading control,α-Tubulin. WT : FMRP=1.00±0.10; GABABR1=1.00±0.06; GABABR2=1.00±0.08. Fmr1 KO : FMRP=0.03±0.01; GABABR1=1.27±0.08; GABABR2=1.54±0.17. Experiment was repeated three times. Significance determined by Student's t-test. Values represent mean±s.e.m. Uncropped versions of qPCR gel, with size markers, are available in Supplementary Fig. 7d. Uncropped version of western blots, with size markers are available in Supplementary Fig. 7b.

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