Journal: The Journal of Biological Chemistry

Article Title: ?-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine *

doi: 10.1074/jbc.M110.127613

Figure Lengend Snippet: GST binding assays in rat intestine. A, schematic diagram of AP-2 complex indicates the known site of CFTR engagement to the μ2 subunit and binding site for Dab2 (DPF) to the α ear appendage. B, schematic map of binding domains in mouse Dab-2. Sequence of amino acids 290–303 of Dab2 indicates mutagenesis sites (black arrows). GST or GST-Dab2 (601–678), Dab2 (1–205), Dab2 (1–368), Dab2 (1–368Δ), and GST-αc-adaptin appendage (250–500 μg) were bound to 60 μl of glutathione-Sepharose bead slurry and incubated with 6.06 μg/ml rat jejunum lysate overnight at 4 °C. The samples were centrifuged to recover the pellet. 20 μg of rat jejunum lysate and one-fourth of each pellet were resolved on SDS-PAGE gels and stained with Coomassie Blue (C–E) or transferred to polyvinylidene difluoride nitrocellulose (F–H). F, Western blots (WB) show binding of GST-Dab2 (601–768) to myosin VI. G, no binding to CFTR is detected with GST-Dab2 1–205 (PTB domain), Dab2 (1–368), or Dab2 (1–368Δ), but GST-αc-adaptin appendage binds robustly to CFTR. H, GST-Dab2 (1–368) and Dab2 (1–368Δ) binding to α-AP-2. The blots were probed with anti-myosin VI, α-AP-2, and CFTR antibodies. The positions of the molecular mass standards (kDa) are indicated.

Article Snippet: Reagents and Antibodies The following antibodies were used in this study: CFTR (M3A7) (Chemicon International), CFTR 217 (Cystic Fibrosis Foundation Therapeutics), alkaline phosphatase (BYA1191) (Accurate Chemicals), AME 4991 CFTR(6), AP-2 α subunit monoclonal antibody (100/2) (Dr. Linton Traub, University Pittsburgh School of Medicine), α-Adaptin 1/2 (Santa Cruz Biotechnology, Inc.) Dab2 (H-110) (Santa Cruz Biotechnology, Inc.), clathrin monoclonal antibody (X-22) (Calbiochem, EMD Division Inc.), myosin 6 (KA-15), Dab2, p96 (BD Transduction Laboratories), rhodamine phalloidin (Invitrogen), fluorescein isothiocyanate-conjugated anti-rabbit and anti-mouse secondary (Invitrogen), Cy-3-conjugated anti-mouse secondary (Jackson Immuno Research Laboratories), and horseradish peroxidase-conjugated secondary antibodies (BD Bioscience).

Techniques: Binding Assay, Sequencing, Mutagenesis, Incubation, SDS Page, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling

doi: 10.1007/s00018-025-05694-w

Figure Lengend Snippet:

Article Snippet: Mouse anti-AP2α1 (α-adaptin 1) (C-5) , Santa Cruz Biotechnology , Cat#sc-398024.

Techniques: Marker, Recombinant, Lysis, Blocking Assay, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Derivative Assay, Plasmid Preparation, Software, Imaging

Journal: Scientific Reports

Article Title: Leukemia Inhibitory Factor-Receptor is Dispensable for Prenatal Testis Development but is Required in Sertoli cells for Normal Spermatogenesis in Mice

doi: 10.1038/s41598-018-30011-w

Figure Lengend Snippet: Details of antibodies used for immunohistochemistry.

Article Snippet: HSD3b , Citrate pH 6 , Santa Cruz Biotechnology Germany , sc30820 , 1:2000 , Santa Cruz Biotechnology Heidelberg, Germany , sc-2961 , 1:200 , ChαG HRP Tyramide.

Techniques: Immunohistochemistry, Plasmid Preparation

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