Journal: Cell reports

Article Title: Sustained AMPAR expression in CA3 neurons may mediate neuroprotection following FLASH-RT

doi: 10.1016/j.celrep.2026.117670

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Article Snippet: Guinea pig anti-GluN1/Grin1 , Alomone , AGC-001-GP; RRID: AB_2756610.

Techniques:

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: TSG101 knockdown selectively alters glutamate receptor surface expression. (A) Representative immunoblots showing total and surface-biotinylated NMDA receptor subunits GluN1 and GluN2A in HEK293 cells transfected with scramble shRNA (control) or shTSG101. Surface proteins were isolated by sulfo-NHS-SS-biotin-based biotinylation and analysed by immunoblotting. GAPDH served as a loading control for total lysate fractions and was absent from biotinylated fractions. (B–C) Quantification of surface GluN1 (B) and GluN2A (C) normalised to total receptor levels and expressed as fold change relative to scramble shRNA. TSG101 knockdown significantly increased the surface abundance of both NMDA receptor subunits. (D) Representative immunoblots showing total and surface-biotinylated AMPA receptor subunits GluA1 and GluA2. (E–F) Quantification of surface GluA1 (E) and GluA2 (F) normalised to total receptor levels. TSG101 knockdown increased GluA1 surface expression; GluA2 surface levels were not significantly altered. (G) Representative immunoblots showing total and surface-biotinylated GluK2. (H) Quantification of GluK2 surface expression normalised to total protein, showing increased surface abundance following TSG101 knockdown. Data are presented as mean ± SEM (n = 3 independent experiments). Unpaired two-tailed Student’s t-test; *p < 0.05, **p < 0.01; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Knockdown, Expressing, Western Blot, Transfection, shRNA, Control, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: ESCRT modulation alters surface localisation of glutamate receptor subunits in HEK293 cells. (A) Representative immunocytochemical images showing surface staining of GluN1 (red) and GluN2A (green) in HEK293 cells under control conditions, following shTSG101, or expression of dominant-negative VPS4a (DN-VPS4a). Merged images include nuclear staining (blue). Scale bar: 20 µm. (B) Quantification of surface GluN1 and GluN2A fluorescence intensity normalised to control. Both shTSG101 and DN-VPS4a significantly altered surface expression of GluN1 and GluN2A. (C–E) Representative images of surface GluA1 (C), GluA2 (D), and GluK2 (E) staining under control, shTSG101, and DN-VPS4a conditions. Scale bars: 20 µm. (F) Quantification of surface GluA1 fluorescence intensity. Both shTSG101 and DN-VPS4a significantly increased GluA1 surface expression. (G) Quantification of surface GluA2 fluorescence intensity. Surface GluA2 showed modest or non-significant changes depending on condition. (H) Quantification of surface GluK2 fluorescence intensity. Both shTSG101 and DN-VPS4a significantly altered GluK2 surface expression. Data are presented as mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Staining, Control, Expressing, Dominant Negative Mutation, Fluorescence

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: NMDA receptor surface expression increases upon ESCRT pathway disruption in primary cortical neurons. (A–C) Representative immunoblots and quantification showing increased surface/total ratios of GluN1 and GluN2A in primary cortical neurons (DIV14) following shTSG101 transduction. β-actin served as a loading control. (D–F) Representative immunoblots and quantification showing increased GluN1 and GluN2A surface/total ratios following DN-VPS4a expression. (G–H) Representative immunocytochemical images and quantification of surface GluN1 and GluN2A under non-permeabilised conditions in neurons expressing scramble shRNA (control) or shTSG101. EGFP indicates transduction (green). Scale bars: 20 µm. (I–L) Representative immunocytochemical images and quantification of surface GluN1 and GluN2A in neurons expressing control vector or DN-VPS4a. Scale bars: 20 µm. Data are presented as mean ± SEM. For biochemical assays, n = 3 independent experiments; for immunocytochemistry, n = 6–8 neurons per condition from 3 independent cultures. One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Expressing, Disruption, Western Blot, Transduction, Control, shRNA, Plasmid Preparation, Immunocytochemistry

Journal: bioRxiv

Article Title: Distinct Endosomal Sorting Complexes Required for Transport Components Differentially Regulate Glutamate and Gamma-Aminobutyric Acid Receptor Surface Expression

doi: 10.64898/2026.06.17.732891

Figure Lengend Snippet: GluN1 redistributes across early, recycling, and late endosomes following ESCRT disruption in primary hippocampal neurons. (A–C) Representative dendritic images showing GluN1 (green) and the early endosome marker EEA1 (red) in control neurons (A), shTSG101-expressing neurons (B), and DN-VPS4a-expressing neurons (C). Merged images with single-channel views and line-scan intensity profiles of GluN1 and EEA1 along dendrites are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (D–F) Representative dendritic images showing GluN1 (green) and the recycling endosome marker Rab11 (red) in control neurons (D), shTSG101-expressing neurons (E), and DN-VPS4a-expressing neurons (F). Line-scan intensity profiles are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (G–I) Representative dendritic images showing GluN1 (green) and the late endosome marker Rab7 (red) in control neurons (G), shTSG101-expressing neurons (H), and DN-VPS4a-expressing neurons (I). Line-scan intensity profiles are shown below each condition. Scale bars: 20 µm (overview), 5 µm (insets). (J–L) Quantification of Pearson’s correlation coefficient (R) for GluN1 colocalisation with EEA1 (J), Rab11 (K), and Rab7 (L) under control, shTSG101, and DN-VPS4a conditions. (M–O) Quantification of Manders’ tM2 values representing the fraction of GluN1 signal within EEA1-positive (M), Rab11-positive (N), and Rab7-positive (O) endosomes. (P) Quantitative summary table of GluN1 colocalisation changes with early (EEA1), recycling (Rab11), and late (Rab7) endosomal markers following shTSG101 or DN-VPS4a. Data represent 3 biological replicates, n = 6 dendrites per condition. All data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test; *p < 0.05, **p < 0.01, ****p < 0.0001; ns, not significant.

Article Snippet: Membranes were blocked in 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and incubated with primary antibodies against GluN1 (mouse, 1A4B10, Synaptic Systems), GluN2A (rabbit, AB1555P, Synaptic Systems), GluA1 (rabbit, 182003, Synaptic Systems), GluA2 (rabbit, 182103, Synaptic Systems), GluK2 (mouse, 1A2B3C, Synaptic Systems), GABRG2 (rabbit, AB_223344, Abcam), GABABR1 (guinea pig, AB_11234, Abcam), TSG101 (rabbit, Ab30871, Abcam), VPS4a (mouse, SAB4200215, Sigma-Aldrich), EGFR (rabbit, AB52894, Abcam), ubiquitin (mouse, P4D1, Sigma-Aldrich; rabbit, U5379, Sigma-Aldrich), and β-actin (mouse, AC-15, Sigma-Aldrich).

Techniques: Disruption, Marker, Control, Expressing