GW182 Antibody

Western Blot: GW182 Antibody [NBP1-57134] -

Western Blot: GW182 Antibody [NBP1-57134] - Transient increase in GW182‐Ago2 interaction & S387 phosphorylation in response to NMDAR stimulationTransient increase in Ago2‐GW182 interaction. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, & lysates were prepared 0, 3, 6, 10, 20 min after NMDA washout & immunoprecipitated with Ago2 antibodies or control IgG. Proteins were detected by Western blotting. The inputs are shown in (B). Graph shows quantification of Ago2‐GW182 interaction, normalised to vehicle control; n = 4. **P < 0.01, ***P < 0.001; one‐way ANOVA, Bonferroni post hoc test. Mean ± SEM.Transient increase in S387 phosphorylation & Akt activation. The same lysates from (A) (1% of input) were analysed by Western blotting using antibodies against pS387 Ago2, Ago2, pS473 Akt, Akt, GW182 & GAPDH as a loading control. Graphs show quantification of pS387 Ago2 levels normalised to total Ago2 (top) & pS473 Akt normalised to total Akt (bottom); n = 4. *P < 0.05; two‐way ANOVA, Bonferroni post hoc test. Mean ± SEM.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29712715), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GW182 Antibody [NBP1-57134] -

Western Blot: GW182 Antibody [NBP1-57134] - NMDA‐induced increase in interaction with GW182 is caused by Ago2 phosphorylation at S387Ago2 S387A mutation blocks, & S387D mutation occludes NMDA‐induced increases in GW182 & DDX6 interactions. Cortical neurons were transfected with molecular replacement constructs expressing Ago2 shRNA plus shRNA‐resistant GFP‐Ago2 (WT, S387A or S387D). Lysates were prepared 10 min after NMDA washout, & GFP‐Ago2 complexes were precipitated using GFP‐trap beads. Bound proteins were detected by Western blotting using GFP, GW182, MOV10, DDX6 or PICK1 antibodies as shown. Graphs show quantification of GFP‐Ago2 interactions, normalised to untreated WT condition; n = 5. *P < 0.05, **P < 0.01; two‐way ANOVA, Bonferroni post hoc test. Mean ± SEM.Ago2 S387A mutation blocks, & S387D mutation occludes NMDA‐induced increase in GW182 co‐localisation in neuronal dendrites. Cortical neurons were transfected with molecular replacement constructs expressing Ago2 shRNA plus shRNA‐resistant GFP‐Ago2 (WT, S387A or S387D), fixed 10 min after NMDA washout, permeabilised & stained with GW182 & GFP antibodies. Graph shows Pearson's co‐localisation coefficients; n = 4 independent experiments (11 cells per condition). *P < 0.05, **P < 0.01; two‐way ANOVA, Bonferroni post hoc test. Scale bar = 10 μm. Mean ± SEM.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29712715), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: GW182 Antibody [NBP1-57134] -

Western Blot: GW182 Antibody [NBP1-57134] - Ago2 association with GW182 in neuronal dendrites increases in response to NMDAR stimulationEndogenous Ago2‐GW182 & Ago2‐DDX6 interactions increase in response to NMDAR stimulation. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, & lysates were prepared 10 min after NMDA washout & immunoprecipitated with Ago2 antibodies. Proteins were detected by Western blotting. Graph shows quantification of Ago2‐GW182 interaction, normalised to vehicle control; n = 5. *P < 0.05; ***P < 0.001; t‐test; mean ± SEM.Analysis of endogenous Ago2‐GW182 co‐localisation in cortical neuronal cultures. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, fixed 10 min after NMDA washout, permeabilised & co‐stained with Ago2 & GW182 antibodies. Representative whole‐cell images are shown. Scale bar = 50 μm.Endogenous GW182‐Ago2 co‐localisation increases in response to NMDAR stimulation in neuronal dendrites. Images show dendrites taken from boxed region in (B), above. Graph shows Pearson's co‐localisation coefficients; n = 4 independent experiments (18–24 cells per condition). *P < 0.05, t‐test. Scale bar = 10 μm. Mean ± SEM.Line‐scan analyses of Ago2 & GW182 fluorescence intensities in control & NMDA‐stimulated dendrites shown in (C).NMDAR stimulation has no effect on endogenous Ago2‐GW182 co‐localisation in neuronal cell bodies. Images show cell bodies taken from boxed region in (B). Graph shows Pearson's co‐localisation coefficients; n = 4 independent experiments (18–20 cells per condition), t‐test. Scale bar = 10 μm. Mean ± SEM.Source data are available online for this figure. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29712715), licensed under a CC-BY license. Not internally tested by Novus Biologicals.