anti ap2a1  (Proteintech)

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A-B, Volcano plot (A) and GO term enrichment analysis (STRING) (B) of the proteins physically associated with miP-PSTPIP2 (versus rabbit IgGs; CTL) in human endothelial cells treated with IL-1β; n=3 independent cell batches. The dashed lines mark the significance threshold (P value: 0.05). C, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells. Nuclei were stained with DAPI. Scale bar: 10 µm D, Co-immunoprecipitation (IP) of miP-PSTPIP2 and β-actin or DAAM1 from human endothelial cell lysates. Similar results were obtained using 2 additional cell batches. E-F, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and caveoilin-1 (CAV1) (E) or Clathrin Heavy Chain (CHC) (F) in human endothelial cells; Scale bar: 10 µm. Similar results were obtained in 3 additional cell batches. G, Co-immunoprecipitation (IP) of miP-PSTPIP2 and CAV1 or CHC from human endothelial cell lysates. Similar results were obtained using 3 additional cell batches. H, Representative confocal images of AP2A1 and miP-PSTPIP2 in human endothelial cells; Scale bar: 50 µm. Similar results were obtained in 3 additional cell batches.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: AP2A1 (1:1000, Novus Biologics, Centennial, United States, NB600-1545), AP2M1 (1:1000, Cell Signalling, Danver, United States, #68196), Arp2 (1:2000, Proteintech, Rosemont, United States, 10922-1-AP), Arp3 (1:1000, Abcam, Cambridge, United Kingdom, ab49671), β-actin (1:1000, Sigma-Aldrich, Burlington, United States, A1978), CAV1 (1:1000, Cell Signaling, #3238), CHC (1:2000, BD Transduction, Franklin Lakes, United States, 610499), Cortactin (1:10000, Abcam, ab81208), DAAM1 (1:2000, Proteintech, 67287-1-Ig), GAPDH (1:300, Millipore, Burlington, United States, MAB374), LDLR (1:2000, Invitrogen, Waltham, United States, PA5-46985), Phospho-AP2M1 (1:1000, Cell Signaling, #7399), RhoA (1:1000, Santa Cruz, Dallas, United States, sc-179) and VASP (1:1000, BD Transduction, V40020).

Techniques: Staining, Immunoprecipitation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Western blot analysis and quantification of clathrin heavy chain (CHC), low-density lipoprotein receptor (LDLR), Caveolin-1 (CAV1), AP-2 complex subunit alpha-1 (AP2A1), phospho (p-AP2M1) and total AP-2 complex subunit mu (AP2M1) expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches. C, Flow cytometry analysis of surface LDLR expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL). Data are shown as mean fluorescence intensity (MFI); n=4 independent cell batches. D, Representative Total Internal Reflection Fluorescence (TIRF) microscopy images of miP-PSTPIP2 and transferrin in human endothelial cells starved of serum overnight and subsequently incubated with AF555–transferrin for 1, 5 or 15 minutes. n=3 independent experiments; scale bar: 20 µm. All statistical analyses were performed using unpaired Student’s t-tests.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: AP2A1 (1:1000, Novus Biologics, Centennial, United States, NB600-1545), AP2M1 (1:1000, Cell Signalling, Danver, United States, #68196), Arp2 (1:2000, Proteintech, Rosemont, United States, 10922-1-AP), Arp3 (1:1000, Abcam, Cambridge, United Kingdom, ab49671), β-actin (1:1000, Sigma-Aldrich, Burlington, United States, A1978), CAV1 (1:1000, Cell Signaling, #3238), CHC (1:2000, BD Transduction, Franklin Lakes, United States, 610499), Cortactin (1:10000, Abcam, ab81208), DAAM1 (1:2000, Proteintech, 67287-1-Ig), GAPDH (1:300, Millipore, Burlington, United States, MAB374), LDLR (1:2000, Invitrogen, Waltham, United States, PA5-46985), Phospho-AP2M1 (1:1000, Cell Signaling, #7399), RhoA (1:1000, Santa Cruz, Dallas, United States, sc-179) and VASP (1:1000, BD Transduction, V40020).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Microscopy, Incubation

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A-B, Volcano plot (A) and GO term enrichment analysis (STRING) (B) of the proteins physically associated with miP-PSTPIP2 (versus rabbit IgGs; CTL) in human endothelial cells treated with IL-1β; n=3 independent cell batches. The dashed lines mark the significance threshold (P value: 0.05). C, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells. Nuclei were stained with DAPI. Scale bar: 10 µm D, Co-immunoprecipitation (IP) of miP-PSTPIP2 and β-actin or DAAM1 from human endothelial cell lysates. Similar results were obtained using 2 additional cell batches. E-F, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and caveoilin-1 (CAV1) (E) or Clathrin Heavy Chain (CHC) (F) in human endothelial cells; Scale bar: 10 µm. Similar results were obtained in 3 additional cell batches. G, Co-immunoprecipitation (IP) of miP-PSTPIP2 and CAV1 or CHC from human endothelial cell lysates. Similar results were obtained using 3 additional cell batches. H, Representative confocal images of AP2A1 and miP-PSTPIP2 in human endothelial cells; Scale bar: 50 µm. Similar results were obtained in 3 additional cell batches.

Article Snippet: Primary antibodies against AP2A1 (1:100, Novus Biologics, NB600-1545), miP-PSTPIP2 (5 μg/ml, Eurogentec, Seraing, Belgium) , control rabbit IgG (5 μg/ml, Millipore, NI01) or phalloidin-Alexa Fluor 633 (1:300, Invitrogen, A22284) were diluted in PBS containing 0.5% horse serum and 0.01% Triton X-100, and incubated for 2 hours at room temperature.

Techniques: Staining, Immunoprecipitation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Western blot analysis and quantification of clathrin heavy chain (CHC), low-density lipoprotein receptor (LDLR), Caveolin-1 (CAV1), AP-2 complex subunit alpha-1 (AP2A1), phospho (p-AP2M1) and total AP-2 complex subunit mu (AP2M1) expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches. C, Flow cytometry analysis of surface LDLR expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL). Data are shown as mean fluorescence intensity (MFI); n=4 independent cell batches. D, Representative Total Internal Reflection Fluorescence (TIRF) microscopy images of miP-PSTPIP2 and transferrin in human endothelial cells starved of serum overnight and subsequently incubated with AF555–transferrin for 1, 5 or 15 minutes. n=3 independent experiments; scale bar: 20 µm. All statistical analyses were performed using unpaired Student’s t-tests.

Article Snippet: Primary antibodies against AP2A1 (1:100, Novus Biologics, NB600-1545), miP-PSTPIP2 (5 μg/ml, Eurogentec, Seraing, Belgium) , control rabbit IgG (5 μg/ml, Millipore, NI01) or phalloidin-Alexa Fluor 633 (1:300, Invitrogen, A22284) were diluted in PBS containing 0.5% horse serum and 0.01% Triton X-100, and incubated for 2 hours at room temperature.

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Microscopy, Incubation

Journal: Cellular and Molecular Life Sciences

Article Title: Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion

doi: 10.1007/s00018-023-05072-4

Figure Lengend Snippet:

Article Snippet: AP2A1 Rabbit mAb , Abclonal , Cat#A4403.

Techniques: Purification, Recombinant, Cell Culture, Modification, Saline, Transfection, Plasmid Preparation, Software

Journal: Frontiers in Immunology

Article Title: Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis

doi: 10.3389/fimmu.2023.1250920

Figure Lengend Snippet: Excessive autophagy leads to the internalization of NKG2D. (A) Using RT-PCR technology to examine RNA expression of the main functional receptors on the NK cell surface. And the reduced NKG2D expression on the surface of NK cells was further confirmed by western blotting (B) and flow cytometry (C) . (D, E) Co-IP demonstrated a significant increase in the interaction between NKG2D and LAMP2 after radiation therapy, and inhibiting autophagy likewise attenuated their binding. (F) A reconstructed three-dimensional structured illumination microscopy (3D-SIM) image showing an NK cell. The cell surface is labeled with an antibody against NKG2D (green), and lysosomes are labeled with a specific lysosomal marker (red). The merged image demonstrates the co-localization of NKG2D and lysosomes (arrow) within the NK cell. Scale bar = 3 μm. A-E: n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. *P = 0.0236 NKp30, ***P = 0.0002 NKG2D, **P = 0.0044 NKG2A (A) , ***P < 0.0001 (B) , ***P = 0.0006 (C) , **P = 0.0021, **P = 0.0016, AP2A1; **P = 0.0039, *P = 0.0390, AP2M1; **P = 0.0060, *P = 0.0472, LAMP2; ***P = 0.0009, *P = 0.0116, LC3 (E) .

Article Snippet: All membranes were blocked by Tween-Tris-buffered saline containing 5% non-fat milk for 2 h at room temperature and then incubated with primary antibodies as follows: GAPDH (#ab9485, Abcam), β-tublin (#ab179511, Abcam), AP2A1 (#ab189995, Abcam), AP2M1(#ab75995, Abcam), LAMP2 (#ab13524, Abcam), LC3 (#ab62721, Abcam), NKG2D (#ab36136, Abcam), CXCR3 (#ab288437, Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Functional Assay, Expressing, Western Blot, Flow Cytometry, Co-Immunoprecipitation Assay, Binding Assay, Microscopy, Labeling, Marker