Journal: Traffic (Copenhagen, Denmark)

Article Title: A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

doi: 10.1111/j.1600-0854.2012.01354.x

Figure Lengend Snippet: Figure 1: Myosin Vb tail mutant affects the recycling pathway. M10+22E cells transiently expressing Myosin Vb-GFP alone (A) or together with Rab11-FIP2-mCh (D), M10+YFP cells transiently expressing Myosin Vb tail-CFP (B) and Rab11-FIP2-mCh (C) were fixed with 4% PFA and immunostained for langerin (A and D, DCGM4), Rab11A (A), early endosome (B and C, EEA1). Single labelings (first three images from left to right) and merged images (last right images) are shown. Scale bars represent 5 μm. Quantification of langerin-YFP fluorescence amounts were performed on Average Intensity Projection of 3D stacks using Imag J tools on control cells (E and F, blue column, n = 14 and 16, respectively) and Myo Vb tail-CFP or Rab11-FIP2-mCh expressing cells (E and F, red column, n = 15 each). YFP fluorescence relative intensities are presented as mean + SD. Immunoblotting (G) was used to confirm the levels of langerin (Lang), in M1022E cells (0), overexpressing MyoVb-Tail mutant (MVb), Rab11-FIP2-mCh (FIP2) or cells infected with Rab11Q70L adenoviruses. This was completed by experiments in the presence (CQ, right panel) or absence (0, right panel) of 50 μM chloroquine, as described in section Material and Methods. Proteins were separated by SDS-PAGE in 12% acrylamide gels and blotted onto membranes, with equivalent amounts of protein being loaded in each lane. The membranes were also probed for β-tubulin (βTub), as a control. Blots are representative of three independent experiments.

Article Snippet: The following antibodies were used as secondary antibodies: Cy3-coupled anti-mouse IgG for TfR and 6C4; Cy3-coupled anti-rabbit IgG for Rab11, Cy3-coupled antigoat for EEA1 (Santa Cruz).

Techniques: Mutagenesis, Expressing, Control, Western Blot, Infection, SDS Page

Journal: Human mutation

Article Title: Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function.

doi: 10.1002/humu.23694

Figure Lengend Snippet: FIGURE 2 PPP1R21 co-localizes with the main early endosome protein EEA1 but not Golgi proteins and PPP1R21 staining is absent in fibroblasts obtained from patient 3_V:4 who is homozygous for c.1607dupT p.(Leu536Phefs*7). (A) In order to determine the entity of PPP1R21 positive vesicles, we performed co-localization studies using Golgi marker GM130 (mouse, ab169276, Abcam, USA) and 58K (ab27043 mouse, Abcam, USA) as well as EEA1 antibody to mark the early endosome. While no co-localization with Golgi markers was observed (upper panels), PPP1R21 (rabbit, HPA036792, Atlas antibodies, Sweden) nearly completely co-localized with EEA1 (lower panel). Scale bar 20 𝜇m. (B) Using two different PPP1R21 antibodies, HPA036792 (ab1, antibody epitope is represented by aminoacid aa161-256) and HPA 036791 (ab2, antibody epitope is represented by aminoacid aa572-666), we found complete loss of the vesicular staining pattern in PPP1R21 mutant fibroblasts we previously observed in control cells while no difference regarding EEA1 staining was detected between PPP1R21 mutant and control fibroblasts. Scale bar: 20 𝜇m

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense REHMAN ET AL. 277 F IGURE 2 PPP1R21 co-localizes with themain early endosome protein EEA1 but not Golgi proteins and PPP1R21 staining is absent in fibroblasts obtained from patient 3_V:4 who is homozygous for c.1607dupT p.(Leu536Phefs*7). (A) In order to determine the entity of PPP1R21 positive vesicles, we performed co-localization studies using Golgi marker GM130 (mouse, ab169276, Abcam, USA) and 58K (ab27043mouse, Abcam, USA) as well as EEA1 antibody tomark the early endosome.While no co-localization with Golgi markers was observed (upper panels), PPP1R21 (rabbit, HPA036792, Atlas antibodies, Sweden) nearly completely co-localized with EEA1 (lower panel).

Techniques: Staining, Marker, Mutagenesis, Control

Journal: Journal of Virology

Article Title: Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

doi: 10.1128/jvi.01338-09

Figure Lengend Snippet: FIG. 8. Localization of gH and MAb 206 relative to EEA1 and Vps4 in fibroblasts in vitro. HELFs inoculated with pOka were mock treated (A to D, K to N) or antibody treated (F to I, P to S) for 48 h, fixed, permeabilized, stained, and examined by confocal microscopy. Uninfected HELFs were mock treated (E and O) or antibody treated (J and T) for 48 h, fixed, permeabilized, stained, and examined. Arrowheads highlight colocalization. (A) gH, red; EEA1, blue; TGN46, green; nuclei, gold. (B) MAb 206, red; EEA1, blue; TGN46, green; nuclei, gold. (C) gH, red; Vps4, blue; TGN46, green; nuclei, gold. (D) MAb 206, red; Vps4, blue; TGN46, green; nuclei, gold. Bar, 5 m.

Article Snippet: Cellular localization of VZV proteins was performed using primary antibodies to VZV proteins gH (SG3 monoclonal mouse anti-gH; Biodesign, Saco, ME), ORF23 (rabbit polyclonal) (7), and gE (rabbit polyclonal) (25) and to cellular proteins TGN46 (AHP500 polyclonal sheep anti-TGN46; AbD Serotec, Oxford, United Kingdom), early endosome antigen 1 (EEA1) (NB300-502 rabbit anti-EEA1; Novus Biologicals, Littleton, CO), and vacuolar protein sorting 4 (Vps4) (sc-32922 rabbit anti-Vps4; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: In Vitro, Staining, Confocal Microscopy