Journal: Cell Structure and Function

Article Title: Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic β-cell Line

doi: 10.1247/csf.20020

Figure Lengend Snippet: GluR2 and GluR3 is expressed in MIN6 cells. (A) Recognition site for anti-extracellular or -intracellular GluR2 or GluR3 antibody used in the study. (B) Western blotting analyses using anti-extracellular GluR2 (upper panel) or GluR3 (middle panel) or anti-intracellular GluR2/3 (bottom panel) antibodies. Membrane fractions from mouse brain (mouse brain, 10 μg) or MIN6 cell membranes (MIN6, 80 μg) were analyzed. (C) Co-localization of GluR2/3 (labeled using anti-intracellular GluR2/3) with clathrin heavy chain (CHC, upper panel) or AP2 (middle panel) in MIN6 cells. An immunofluorescence image without primary antibody is shown as a negative control (bottom panels). Scale bar, 20 μm. (D) Co-localization measurement of GluR2/3 and CHC or AP2 in MIN6 cells. The data shown are the mean±S.E.M. from 20 cells in three independent experiments. In each sample, co-localization was determined in three randomly selected areas per cell (21 μm 2 ) using overlay images.

Article Snippet: A mouse monoclonal antibody against α-adaptin (AP2) (cat# CP46) was purchased from Merck KGaA.

Techniques: Western Blot, Membrane, Labeling, Immunofluorescence, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors *

doi: 10.1074/jbc.M113.535211

Figure Lengend Snippet: MHCI endocytosis depends on clathrin and AP-2. A, chemical structures of Pitstop-2, Pitstop 2–100, Pitnot-2, and Pitnot-2-100. B, IC50 values of Pitstop-2- or Pitstop-2-100-mediated interference with clathrin TD complex formation determined by ELISA. C, internalization of transferrin-Alexa 488 into HeLa cells is inhibited by Pitstop-2 or Pitstop-2-100 (30 μm). The amount of transferrin-Alexa 488 internalized into DMSO-treated cells was set to 100%. D, MHCI internalization into HeLa cells is inhibited by Pitstop-2 or the related compound Pitstop-2-100 (30 μm) but not by two different non-clathrin-binding control compounds. Scale bar, 10 μm. E, representative images of MHCI internalization into CHC- or AP-2(μ)-depleted HeLa cells. Cells were treated with either DMSO or Pitstop-2. Scale bar, 10 μm. F, quantification of data shown in D (n = 3 independent experiments). The amount of MHCI internalized into DMSO-treated cells was set to 100%. G, quantification of MHCI internalization into clathrin (CHC k.d.)- or AP-2(μ)-depleted (μ2 k.d.) cells treated with DMSO or Pitstop-2 as shown in E. The amount of MHCI internalized into DMSO-treated control cells was set to 100% (n = 3 independent experiments; *, p < 0.05; **, p < 0.005; ***, p < 0.0005). H, representative images of MHCI surface levels of CHC- or AP-2(μ)-depleted HeLa cells. Cells were treated with either DMSO or Pitstop-2. Scale bar, 10 μm. I, quantification of clathrin (CHC) or AP-2(α) levels in cells treated with siRNA against AP-2(μ) or CHC or mock transfected (n = 3 independent experiments).

Article Snippet: For Western blot experiments, antibodies were used against clathrin heavy chain (TD1, homebrew), AP-1 (γ1-adaptin, BD Transduction, 610386), AP-2 (α-adaptin AC1M11, Dianova, MA3-061), AP-3 (μ3-adaptin p47, BD Transduction, 610901), EGF receptor (D38B1, Cell Signaling, 4267S), and β-actin (ac-15, Sigma, A-5441).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors *

doi: 10.1074/jbc.M113.535211

Figure Lengend Snippet: Pitstop-2-mediated inhibition of CCP dynamics has no major effects on general plasma membrane mobility. A, time lapse series of cells stably expressing AP-2(σ2)-eGFP were collected after clathrin HC depletion or mock treatment using live cell TIRF microscopy. Cells were imaged for 2 min at 37 °C in presence of 30 μm Pitstop-2 or DMSO (0.1%). AP-2(σ2)-eGFP distribution is unaffected by clathrin HC knockdown (CHC k.d.) or application of Pitstop-2, respectively. Kymographs depict stalled AP-2(σ2)-eGFP dynamics in absence of clathrin HC or after Pitstop-2-mediated inhibition of clathrin TD. Scale bar, 10 μm. B, FRAP analysis of HeLa expressing GPI-eGFP and either left untreated or treated with 0.1% DMSO or with 30 μm of the indicated compounds (Pitstop-2, Pitstop-2-100, Pitnot-2, or Pitnot-2-100; see Fig. 1A). 60 s after bleaching, Pitstop-2-treated cells show similar recovery to DMSO-treated controls. Scale bar, 10 μm. C, quantitative analysis of fluorescence recovery as shown in B. Graphs represent the relative fluorescence recovery normalized to the fluorescent intensity before bleaching (n = 3 independent experiments; *, p < 0.05).

Article Snippet: For Western blot experiments, antibodies were used against clathrin heavy chain (TD1, homebrew), AP-1 (γ1-adaptin, BD Transduction, 610386), AP-2 (α-adaptin AC1M11, Dianova, MA3-061), AP-3 (μ3-adaptin p47, BD Transduction, 610901), EGF receptor (D38B1, Cell Signaling, 4267S), and β-actin (ac-15, Sigma, A-5441).

Techniques: Inhibition, Stable Transfection, Expressing, Microscopy, Fluorescence