Journal: Genes & Development

Article Title: Ephrin-B2 controls PDGFRβ internalization and signaling

doi: 10.1101/gad.224089.113

Figure Lengend Snippet: Eph–ephrin binding triggers PDGFRβ internalization. ( A ) Indirect immunofluorescence of PDGFRβ (green) in cultured murine VSMCs stimulated with IgG/Fc, ephrin-B2/Fc, or EphB4/Fc for 2 h, as indicated. (Red) Actin (phalloidin); (blue) nuclei (DAPI). Note accumulation of PDGFRβ in perinuclear structures resembling vesicles after EphB4/Fc but not ephrin-B2/Fc treatment. The panels at the right show higher magnification of the insets . ( B ) Western blot showing reduction of biotinylated (surface) ephrin-B2 and PDGFRβ in cultured murine VSMCs treated with EphB4/Fc. Nystatin treatment had a mild effect on EphB4/Fc-induced PDGFRβ and ephrin-B2 internalization. ( C ) Densiometric analysis of biotinylated (surface) PDGFRβ shown in B . P -values were calculated using two-tailed Student's t -test ( n = 3). Error bars indicate SD. ( D ) Immunofluorescence of ephrin-B2 (detected by EphB4/Fc binding; red), PDGFRβ (green), and EEA1 (blue) in murine VSMCs at 0.5 and 2.5 h after EphB4/Fc treatment. Higher magnifications of the insets in the left images are shown in the other panels. Arrowheads indicate colocalization (white) of ephrin-B2, PDGFRβ, and EEA1 in early endosomes; arrows mark ephrin-B2 + and PDGFRβ + but EEA1 − structures. ( E ) Sucrose density gradient centrifugation of VSCMs at 30 min after stimulation with IgG/Fc, EphB4/Fc, or EphB4/Fc+PDFG-B, as indicated. EphB4/Fc triggered redistribution of PDGFRβ from fractions 4–7 into fractions 8–11. Active PDGFRβ (P-PDGFRβ) at 5 min after stimulation with PDGF-B was associated with fractions 8–11. Molecular weight markers (in kilodaltons) are indicated. ( F ) Western blot showing that Erk1/2 and PDGFRβ phosphorylation in PDGF-B-stimulated VSMCs (10 min) was enhanced by pretreatment with EphB4/Fc for the indicated times. Total Erk1/2 and PDGFRβ levels and molecular weight markers (in kilodaltons) are shown. Statistical analysis of p-Erk1/2 is provided in Supplemental Fig. 5C.

Article Snippet: Fractions were analyzed by immunoblot with antibodies detecting caveolin-1 (1:500 dilution; Becton Dickinson, catalog no. 610493), CHC (1:1000; Becton Dickinson, 610500), EEA1 (1:000; Novus Biologicals, NBP1-05962), PDGFRβ (1:1000; R&D Systems, 3169), phospho-PDGFRβ (1:1000; R&D System, 3161), ephrin-B2 (1:1000; Sigma, HAP008999), or VEGFR3 (1:1000; eBioscience, 14-5988).

Techniques: Binding Assay, Immunofluorescence, Cell Culture, Western Blot, Two Tailed Test, Gradient Centrifugation, Molecular Weight, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

doi: 10.1091/mbc.E11-04-0383

Figure Lengend Snippet: Depletion of Myo1E reduces endocytosis and inhibits downstream endocytic trafficking. (A) Western blots and quantification of Myo1E depletion using siRNAs. Depletion of Myo1E in HeLa and SK-MEL-28 cells after 72 h showed a 96 and 90% reduction in Myo1E protein levels, respectively. Sec23 serves as a loading control. (B) ELISA-based transferrin-uptake assay showed a 27 and 36% reduction in internalization of biotinylated transferrin at 4-min in Myo1E-depleted HeLa and SK-MEL-28 cells compared with control, respectively. * p < 0.05 and ** p < 0.01. (C, F) F-actin staining and (D, G) fluorescent transferrin uptake in control and Myo1E-depleted SK-MEL-28 cells, respectively. Ten minutes after internalization, Alexa Fluor 488-transferrin–containing puncta were concentrated at the perinuclear region in the control cells (D). (D, G) By quantifying the total fluorescence of the cell, there is a 51 ± 18% reduction of the internalized transferrin in Myo1E-depleted cells compared with the control cells (cells = 6 and 7 for Myo1E-depleted and control cells, respectively). In addition, the internalized puncta were localized at the cell periphery rather than at the perinuclear region. (I–K) Immunofluorescence shows colocalization of the perinuclear-concentrated Alexa Fluor 488–transferrin puncta and an early endosome marker EEA1 in the control cells. However, in the Myo1E-depleted cells (L–N), the puncta were dispersely localized and did not colocalize with EEA1, which retains its perinuclear localization. Bar, 10 μm.

Article Snippet: The polyclonal rabbit anti-EEA1 antibody (2411) was purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Immunofluorescence, Marker