mouse anti ap2  (Developmental Studies Hybridoma Bank)

Journal: Cell Discovery

Article Title: Clathrin mediates membrane fission and budding by constricting membrane pores

doi: 10.1038/s41421-024-00677-w

Figure Lengend Snippet: a Left: setup drawing. The cell membrane, bath and vesicles are labelled with PH G (green), A655 (red) and FFN511 (cyan), respectively. ICa and capacitance (Cm) are recorded via a whole-cell pipette. Right: sampled ICa and Cm induced by depol 1s . b Confocal images of PH G , A655, and FFN511 (near cell-bottom) showing pre-spot I, II, and III at the XY- (left, ring-shape) and XZ-plane (right, Ω-shape). c PH G fluorescence (F PH ), A655 fluorescence (F 655 ), FFN511 fluorescence (F FFN ), R fs+pre , and sampled confocal images showing depol 1s -induced (triangle) rapid (left), slow (middle) or large-size (right) pre-spot pore closure (pre-close). A reconstructed trace reflecting pre-close (R fs+pre , a down-step at F 655 dimming onset) is also plotted. F PH , F 655 and F FFN were normalized to the baseline. d , e F PH , F 655 , F FFN , R fs+pre and confocal images showing rapid and slow close-fusion ( d ), stay- or shrink-fusion ( e ). R fs+pre : a reconstructed trace reflecting fusion (up-step, at F 655 rising onset) and fusion pore closure (down-step, at F 655 dimming onset). f Left: sampled western blot results of CHC, dynamin 1/2 (Dyn), adaptor protein 2 α subunit (AP2), synaptotagmin 1 (Syt1), syntaxin 1 (Stx1), and actin in chromaffin cell cultures transfected with si-Ctrl or si-CHC. Right: the percentage (mean ± s.e.m., 3 transfections) of the above proteins in cultures transfected with si-CHC (data normalized to the corresponding mean of si-Ctrl). g – i The percentage of pre-spots undergoing pore closure (pre-close%, g ), the percentage of close-fusion in all fusion events (close-fusion%, h ), and FFN511 20%–80% decay time ( i ) in: (1) si-Ctrl (19 cells), si-CHC (21 cells), or si-CHC+CHC (si-CHC transfection plus CHC overexpression, 17 cells); (2) sh-Ctrl (16 cells) or sh-CHC (16 cells, FFN511 not included); and (3) Ctrl (16 cells, no pitstop 2) or pitstop 2 (PST2, 30 μM, bath, 15–30 min; 15 cells). *** P < 0.001 ( t -test). j Sampled FFN511 spot fluorescence (F FFN ) decay in si-Ctrl (dotted) or si-CHC (solid) showing faster release after clathrin knockdown.

Article Snippet: Primary antibodies were diluted in PBS containing 10% donkey serum and incubated with cells for 1 h at 22–24 o C. After several rinses in PBS, cells were incubated with fluorescence-conjugated donkey anti-mouse, anti-sheep, or anti-rabbit IgG (1:1000, Invitrogen) for 2 h at 22–24 o C. Primary antibodies included mouse anti-CHC (1:500, Abcam), mouse anti-AP2 (1:100, ThermoFisher Scientific) and rabbit anti-endophilin 1 (1:200, Invitrogen), rabbit anti-dynamin 1 (1:150, Abcam), mouse anti-SNAP25 (1:500, Synaptic Systems) and mouse anti-syntaxin (1:500, Synaptic Systems).

Techniques: Membrane, Transferring, Fluorescence, Western Blot, Transfection, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: Potential binding proteins of EMCN involved in endocytosis identified through mass spectrometry. A Diagram illustrating the workflow of EMCN immunoprecipitation in HRECs lysate and subsequent identification of potential EMCN-binding proteins using mass spectrometry. B Coomassie blue stained SDS-PAGE gel showing the separation of EMCN binding proteins, which were then excised for mass spectrometric analysis. C Protein clusters involved in endocytosis among EMCN-binding proteins identified by mass spectrometry and analyzed using the STRING database. D List of endocytosis related EMCN binding proteins identified in mass spectrometry. E Validation of the interaction between EMCN and AP2A2 by immunoprecipitation on Western blot. F HRECs overexpressing myc-tagged EMCN were lysed and both AP2 α and β subunits co-immunoprecipitated with EMCN. FT: flowthrough. n = 3

Article Snippet: After 4% PFA fixation at room temperature and permeabilization using 5% serum in PBS with 0.1% Triton X-100, cell was then incubated with goat anti-VEGFR2 (1:200, AF357; R&D Systems), Rabbit anti clathrin (1:200, 4796 T; Cell Signaling Technology) and mouse anti AP2 (1:200, F-12, Santa Cruz) at 4 °C for overnight.

Techniques: Binding Assay, Mass Spectrometry, Immunoprecipitation, Staining, SDS Page, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: Essential role of EMCN in facilitating VEGFR2 and AP2 interaction and clathrin recruitment. A Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in control HRECs with or without VEGF165 (10 ng/ml). Examples of colocalization of VEGFR2 and clathrin HC (white arrowhead), and clathrin (white arrow) were shown in magnified view. Bar = 10 µm B Fraction of VEGFR2 that colocalized with clathrin was quantified by Image J CoJAP plugin. Student t-test was used for the comparison. * P < 0.05, n = 3. C Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in siNT or siEMCN HRECs with VEGF165 (10 ng/ml) stimulation. Examples of colocalization of VEGFR2 and clathrin HC (white arrowheads), and VEGFR2 (white arrow) were shown in magnified view. Bar = 10 µm D Fraction of VEGFR2 that colocalized with clathrin in siNT and siEMCN HRECs was quantified. Student t-test was used for the comparison. ** P < 0.01, n = 3. E Colocalization of clathrin HC (heavy chain) (green) and AP2 (red) were visualized in control in siNT and siEMCN HRECs with VEGF(10 ng/ml) stimulation. Examples of colocalization of clathrin HC and AP2 (white arrowhead) were shown in magnified view. Bar = 10 µm ( F ) Fraction of clathrin that colocalized with AP2 in siNT and siEMCN HRECs with VEGF stimulation were quantified. Student t-test was used for the comparison. P > 0.05, n = 3. G EMCN is required for interaction between VEGFR2 and AP2A2. HRECs were lysed and VEGFR2 that co-immunoprecipitated with AP2A2 in the presence and absence of EMCN was observed. n = 3

Article Snippet: After 4% PFA fixation at room temperature and permeabilization using 5% serum in PBS with 0.1% Triton X-100, cell was then incubated with goat anti-VEGFR2 (1:200, AF357; R&D Systems), Rabbit anti clathrin (1:200, 4796 T; Cell Signaling Technology) and mouse anti AP2 (1:200, F-12, Santa Cruz) at 4 °C for overnight.

Techniques: Comparison, Immunoprecipitation