atto594 red rabbit anti glua1 antibody  (Alomone Labs)

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: ( A - C ) Surface biotinylation was used to isolate endogenous surface (s) GluA1 and sGluA2 in acute striatal slices derived from wildtype (WT) and Lrrk2 G2019S (GS) mice. A ) Representative Western blot images of sGluA1, sGluA2, and actin from dataset quantified in B and C . Scatterplot/bar graphs plot values normalized to WT ± SEM (n = 7 - 8 mice/genotype, 3 slices/mouse). Unpaired t test *p= 0.0492, compared to WT. D ) Confocal image of DARPP32-labeled (white) co-cultured SPN (left) and super-resolution, STED images (right) of dendritic processes. Punctate sGluA1 (magenta), sGluA2 (green) labeling (tagged prior to permeabilization) associates largely, but not completed with PSD95 labeling (blue). E and F ) Violin plots compare surface AMPAR area ( E ) and intensity ( F ) within masks defined by PSD95 labeling in SPNs. Unpaired t test, ****p<0.0001; ***p<0.0001; Mann Whitney test *p=0.04. n= 3 preps and 15 ROIs/genotype.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Derivative Assay, Western Blot, Labeling, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A ) Schematic outlines antibody feeding assay used to monitor GluA1 internalization in wildtype (WT) and Lrrk2 G2019S (GS) corticostriatal co-cultures (DIV16-18) and serves as a key for the colors used to show data. B and C ) Overlay images show labeled surface (s) GluA1 (green mask + red mask) and internalized (i) GluA1 (red mask only) signal contained within DARPP-32 labeled SPNs (shown at a reduced intensity to permit visualization of puncta. Masks were generated in Image J and magnification is shown in B. D - G ) Intensity distribution of green and red labeling along a 60 µm line scan. H ) Quantification of the internalization index of GluA1 receptors in WT and GS SPNs at 0 and 60 min (n = 12 - 16 cells, 3 preps/genotype). Two-way ANOVA (F ( , ) = 9.809, p = 0.0029), post hoc Šidák test **p = 0.0049.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Feeding Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Parkinson’s-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity

doi: 10.1101/2023.10.13.562231

Figure Lengend Snippet: A, C ) Bar graph/scatterplots comparing functional contribution of CP-AMPARs using ratios of EPSCs evoked in the presence/absence of NASPM in D 1 R ( A; Drd1tdTom+ ) and D 2 R ( C; Drd1tdTom- ) SPNs in WT and GS mice (P70 - P90) in acute slices through dorsal striatum. Bars are mean ± SEM (n = 11 cells, 5 - 6 mice/group, unpaired t test *p = 0.0122). Example traces ( B ) show AMPAR currents before (black) and after (lavender) bath application of NASPM (200µM, 10 min). D - G , Superresolution (tau-STED) images ( D, E ) and quantification ( F, G ) of synaptic and extrasynaptic receptors in 21 DIV WT and GS D 1 R SPNs expressing tdTomato (white, Drd1 Cre/+; Ai14 , in D, E, and used to segment D1R SPNs for F, G) and co-cultured with unlabeled cortical neurons of the same genotype. sGluA1 ( D , magenta, STED) and sGluA2 ( E , green, STED) puncta in relation to PSD95 labeled postsynaptic sites (blue, confocal). Circled zones are enlarged in the center panels. F, G ) Bar graph/scatterplots show synaptic ( F , within a mask defined by PSD95) and extrasynaptic ( G , outside a PSD95 mask) sGluA1 (lavender) and sGluA2 (green) nanoclusters in D 1 R SPNs. F : Unpaired t test **p = 0.0369; n = 16 ROIs/genotype). G : Unpaired t test ****p<0.0001; n = 16 ROIs/genotype. H - K ) Examples ( H ) and quantification ( I - K ) of FRAP experiments. (H) Time lapse confocal images pre- and postphotobleaching (dotted circles approximate ROIs) in WT and GS D 1 R SPNs labeled and co-cultured as above. Table ( I ) compares time constant, diffusion (D) and % mobile receptors. D was calculated using: D = 0.25 (r 2 /τ 1/2 ), where r refers to the bleach radius and τ 1/2 to the time constant 46 . Graph ( J ) plots normalized SEP-GluA1 fluorescence recov-ery in WT and GS neurons imaged every 2.5 s. Lighter shading is ± SEM. Two-way RM-ANOVA (F(89, 3115) = 17.79, **p < 0.001, n = 19-20 spines/genotype. Scatterplot ( K ) compares recovery of SEP-GluA1 intensity at T222.5 s time point relative to post-bleach in WT and GS D 1 R SPNs. One-way ANOVA (F(3, 70) = 24.15, p < 0.0001), post hoc Tukey’s multiple comparison test ****p<0.0001.

Article Snippet: Cultured neurons were live-labeled with a direct-conjugated, ATTO-594 (red) rabbit anti-GluA1 antibody (1:100, Alomone AGC-004-AR, RRID:AB_2340944) for 15 min at room temperature.

Techniques: Functional Assay, Expressing, Cell Culture, Labeling, Diffusion-based Assay, Fluorescence, Comparison