Journal: eNeuro

Article Title: Comparative In Vivo Imaging of Retinal Structures in Tree Shrews, Humans, and Mice

doi: 10.1523/ENEURO.0373-23.2024

Figure Lengend Snippet: A list of the primary antibodies tested in tree shew and mouse retinas

Article Snippet: AP2-α , Activating protein-2 , DSHB catalog #3B5 , AB_2313947 , Mouse , 1:200 , Y/Y.

Techniques:

Journal: bioRxiv

Article Title: The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis

doi: 10.1101/2023.01.15.524101

Figure Lengend Snippet: Domain-specific mutations in Amph1 disrupt its ability to form protein-protein complexes. (A) Schematic representation of the structural domains of Amph1 that were used for generating GST-fused N-BAR-lacking mutants either with or without the SH3 domain. Point mutations were introduced within the CLAP domain including FFE to SSR for the α-AP2 binding site; and DLD to HSR and WD to SR for the CHC binding sites as indicated. (B) GST-pull-down experiments were performed using synaptosomal lysates from rat brains and GST-fused Amph1-ΔNBAR mutants. The binding affinity of purified endocytic proteins including endophilin A1, CHC, α-AP2 subunits, and Dyn1 was assessed by immunoblotting. CBB staining was performed in parallel to assess GST-fusion protein levels (arrows). Representative images are displayed. The abnormal separation of α-AP2 subunits may result from the high protein content of the GST-Amph1-ΔSH3 constructs which run at a similar molecular weight. (C-F) Quantification of the binding efficacy of CHC (C), α-AP2 subunits (D), Dyn1 (E), and endophilin A1 (F) by determining relative band intensities normalised to the level of the total GST-fused proteins and WT controls. Bars indicate mean ± SEM. ** p < 0.01, *** p < 0.001, and ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. n = 4 synaptosomal lysates/condition from 4 rat brains.

Article Snippet: The primary antibodies used in this study are as follows: goat endophilin A1 (1:1000; Santa Cruz Biotechnology, cat no. sc-10874), goat CHC (1:250: Santa Cruz Biotechnology, cat no. sc-6579), mouse α-AP2 (1:1000; Sigma Aldrich, cat no. A4325), goat Dyn1 (1:500; Santa Cruz Biotechnology, sc-6402), goat syndapin 1 (1:1000; Santa Cruz Biotechnology, sc-10412), and mouse PSD95 (1:1000; BioLegend, cat no. 810401).

Techniques: Binding Assay, Affinity Purification, Western Blot, Staining, Construct, Molecular Weight

Journal: bioRxiv

Article Title: The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis

doi: 10.1101/2023.01.15.524101

Figure Lengend Snippet: The Amph1-endophilin A1 complex is formed at the motif 301 PPVPP 305 within the PRD and is controlled by the phosphorylation status of Amph1-S293. (A) Schematic representation of the structural domains of Amph1 that were used for generating GST-fused ΔN-BAR- and ΔSH3 mutants. Point mutations were introduced within the PRD domain including substitutions of proline to alanine residues for AVRA, A 1 A 2 VA 3 A 4 , A1A2, and A3A4 mutants, and S293A and S293E respectively. (B) GST-pull-down experiments using synaptosomal lysates from rat brains and GST-fused Amph1-ΔNBAR-ΔSH3 mutants to assess binding affinity to endophilin A1 and other SH3-containing proteins, such as CHC, α-AP2, PSD95, and syndapin 1. CBB staining was performed in parallel to assess GST-fusion protein levels (arrows). Representative images are displayed. The abnormal separation of α-AP2 subunits may result from the high protein content of the Amph1-ΔNBAR-ΔSH3 constructs which run at a similar molecular weight. (C) Quantification of the endophilin binding efficacy by determining relative band intensities normalised to the level of the GST-fused proteins and WT Amph1-ΔNBAR-ΔSH3. Bars indicate mean ± SEM. n = 4 synaptosomal lysates/condition from 4 rat brains, * p < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The primary antibodies used in this study are as follows: goat endophilin A1 (1:1000; Santa Cruz Biotechnology, cat no. sc-10874), goat CHC (1:250: Santa Cruz Biotechnology, cat no. sc-6579), mouse α-AP2 (1:1000; Sigma Aldrich, cat no. A4325), goat Dyn1 (1:500; Santa Cruz Biotechnology, sc-6402), goat syndapin 1 (1:1000; Santa Cruz Biotechnology, sc-10412), and mouse PSD95 (1:1000; BioLegend, cat no. 810401).

Techniques: Binding Assay, Staining, Construct, Molecular Weight

Journal: Cell reports

Article Title: Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

doi: 10.1016/j.celrep.2022.110950

Figure Lengend Snippet: (A–G) HSC3 cells were transfected with non-targeting (siNT) or TFG (siTFG-1 or siTFG-2) siRNAs and used for experiments after 3 days. (A) Cells were lysed, and the lysates were probed by western blotting with antibodies to TFG and α-adaptin ( αAd , loading control). The amount of residual TFG in depleted cells is presented as percent of TFG amount in control (siNT) cells (n = 9 from several experiments). (B) Cells were incubated with 20 ng/mL 125 I-EGF for 15 min at 37°C, and the amount of surface-bound and internalized 125 I-EGF was determined. Mean values with SDs are presented in the bar graph (n = 3). This experiment is representative of three independent experiments. p values were calculated for siTFG-1 and −2 against siNT. ns, p > 0.05. (C) Schematic design of 125 I-EGF recycling/degradation assay. See for details. (D) 125 I-EGF recycling/degradation assay was performed as in (C). Cells were incubated with 20 ng/mL 125 I-EGF for 15 min followed by mild acid wash at 4°C and chase incubation at 37°C for 0, 15, and 120 min. The amount of surface-bound, intracellular, intact 125 I-EGF in the medium and low molecular weight degradation products of 125 I-EGF in the medium were determined and expressed as fractions of total 125 I-EGF in 125 I-EGF-loaded cells. Bar graphs represent mean values with SDs (n = 3). p values for siTFG-1 and −2 against siNT were calculated using one-way ANOVA (Tukey’s multiple comparison test). p values are not shown if they are >0.05. This experiment is representative of three independent experiments. Efficient TFG depletion was determined in parallel cultures and shown in (E). (E) TFG deletion in cell cultures grown in parallel with the experiments presented in (D) and (F). Cells were lysed, and the lysates were probed by western blotting with antibodies to TFG and αAd (loading control). (F) Cells were incubated with Tfn-TxR for 1 hr at 37°C followed by mild acid wash (pH 4.5) to minimize the amount of Tfn-TxR at the cell surface and chased in the presence of excess unlabeled Tfn for 0 or 60 min. The cells were then fixed and stained with DAPI, and 3D images were acquired through 405- (blue, nuclei) and 561-nm (magenta, Tfn-TxR) laser channels. All images are single sections through the middle of the cell of representative 3D images. Scale bars, 10 μm. (G) Quantification of the amount of Tfn-TxR per cell from images exemplified in (F). Bar graph represents mean values with SEMs (n of 7 FOVs). ns, p > 0.05. p values are calculated for siTFG-1 and −2 against siNT using one-way ANOVA (Tukey’s multiple comparison test). This experiment is representative of two independent experiments.

Article Snippet: Monoclonal IgG2a antibody to α-adaptin (α subunit of AP2) (AB_2056321) was from ThermoFisher (Waltham, MA).

Techniques: Transfection, Western Blot, Control, Incubation, Degradation Assay, Molecular Weight, Comparison, Staining

Journal: Cell reports

Article Title: Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR

doi: 10.1016/j.celrep.2022.110950

Figure Lengend Snippet:

Article Snippet: Monoclonal IgG2a antibody to α-adaptin (α subunit of AP2) (AB_2056321) was from ThermoFisher (Waltham, MA).

Techniques: Transduction, Recombinant, Mass Spectrometry, Sequencing, Software