Journal: Virology Journal

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

doi: 10.1186/1743-422X-11-40

Figure Lengend Snippet: Effect of dominant negative Rab GTPases on ABLV G-mediated viral entry. HEK293T cells expressing dsRed-tagged WT and DN Rab5, Rab7, and Rab11were infected with maxGFP encoding rVSV that express ABLVp G, ABLVs G, or VSV G at a MOI = 3 for 8 hrs or with rVSV that expresses EboGP at a MOI = 15 for 20 hrs and then analyzed as described in Figure . Infection of all cells was assessed. Under these conditions, the chosen MOIs yielded 40-50% virus-infected cells in controls. Results are expressed as percent virus-infected cells relative to that of WT Rab controls and represent 3 independent experiments; error bars are SEM. (A) Rab5. (B) Rab7. (C) Rab11. **, p < 0.0001; *, p < 0.005.

Article Snippet: DsRed-tagged Rab7 wild-type (WT) (plasmid #12661) and DN (T22N; plasmid #12662), DsRed-tagged Rab11 WT (plasmid #12679) and DN (S25N; plasmid #12680) [ ], and mRFP-tagged Rab5 WT (plasmid #14437) [ ] were purchased from Addgene, Cambridge, MA.

Techniques: Dominant Negative Mutation, Expressing, Infection, Virus

Journal: Biochimica et biophysica acta

Article Title: SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover.

doi: 10.1016/j.bbamcr.2010.09.008

Figure Lengend Snippet: Fig. 5. Inhibition of SNARE-mediated membrane traffic inhibits Src trafficking from a Rab11 compartment. CHO-E329Q cells were mock treated (Wild-type NSF; A–C, G–I) or induced with doxycycline (E329Q-NSF; D–F, J–L) for 9 h and serum-starved for the last hour, before being lifted, and plated on FN for the indicated times. Cells were fixed in ice-cold 0.1% Triton X-100, 2% paraformaldehyde and stained with anti-Src (red; A, D, G, and J) and anti-Rab11 (green; B, E, H, and K). Arrowheads point to Src-Rab11-containing compartment in WT-NSF cells (I) and E329Q-NSF cells (L). Note the increased amount of Src in Rab11-containing compartment in L. Scale bar=10 μm. (M) Cells from the above experiments, as well as untreated cells, and cells where membrane traffic was inhibited with truncated SNAP23 (SN23CΔ9) and dominant-negative Rab11 (Rab11S25N) were visually scored as having primarily perinuclear Src staining (light grey bars), as seen in panel A, or peripheral Src staining (dark grey bars), as seen in panel G. Results are the means±SEM of at least 3 separate experiments in which at least 50 cells were scored.

Article Snippet: Antibodies were obtained against NSF from Stressgen Biotechnologies (Victoria, BC), Rab11, phosphoTyr397-FAK and mouse anti-FAK (F15020) from BD Biosciences (Mississauga, ON), rabbit anti-FAK from Abcam (ab40794; Cambridge, MA), phosphoTyr576-FAK from Novus Biologicals (Littleton, CO), phosphoTyr925-FAK and phosphoSer473Akt from Cell Signaling Technology, Inc. (Boston, MA), phosphoTyr418Src, phosphoTyr861-FAK and Src from Applied Biological Materials, Inc. (Richmond, BC), α-tubulin from Invitrogen, Co. (A11126; Mississauga, ON), and Rac from Cytoskeleton, Inc. (Denver, CO).

Techniques: Inhibition, Membrane, Staining, Dominant Negative Mutation

Journal: Nature Communications

Article Title: PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis

doi: 10.1038/ncomms13546

Figure Lengend Snippet: ( a ) Time-course analysis of recycled active α5β1 integrin in siCTL ECs versus siRAB11A ECs or siRAB11B ECs. RAB11B, but not RAB11A, silencing significantly impairs active α5β1 integrin recycling. Data are mean±s.e.m., n =3 independent experiments (two technical replicates for each experiment). ( b ) Confocal microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). SNAKA51 + active α5β1 integrin localizes in fibrillar adhesion in siCTL and in siRAB11A, but not in siRAB11B ECs in which it accumulates in perinuclear punctae. The number of active α5β1 integrin-containing adhesions per 100 μm 2 of cell area was quantified in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( c ) Confocal xz sectioning microscopy analysis of anti-active α5β1 integrin mAb SNAKA51 localization (red) in living confluent ECs (20 min of incubation). Quantitative analysis of apico-basal mean intensity ratio reveals that SNAKA51 + active α5β1 integrin localizes on the basolateral surface of siCTL and siRAB11A, but not of siRAB11B ECs, where it redistributes all around the cell surface. Data are mean values±s.e.m., n =20 cells per condition pooled from two independent experiments. ( d ) Confocal microscopy analysis of IST9 mAb + endogenous cellular ED-A FN (green) in confluent ECs. ED-A FN polymerizes into a fibrillar network in siCTL and siRAB11A, but not in siRAB11B ECs, where it accumulates in a perinuclear compartment. Relative amount of fibrillary ED-A FN area was calculated in siCTL, siRAB11A and siRAB11B ECs. Data are mean values±s.e.m., n =20 cells per condition pooled from 2 independent experiments. ( e ) Western blot analysis of soluble ED-A FN released by confluent ECs seeded on Transwell inserts. An equal percentage of apical and basolateral volumes of medium were collected after 72 h of culture from different wells of siCTL or siRAB11B ECs. Equal amounts of rabbit IgG were exogenously added to samples (spike normalization) for loading control purposes. Quantification of the ratio between apical or basolateral amount of ED-A FN released by siCTL over siRAB11B ECs. RAB11B silencing much more severely impairs basolateral than apical ED-A FN secretion. Data are mean±s.e.m., n =6 wells per condition pooled from three independent experiments. Scale bar, 50 μm ( d ), 20 μm ( b ), 5μm ( c ). ** P <0.01; *** P <0.001; Student's t -test.

Article Snippet: Rabbit polyclonal anti-RAB11A (TA324158) and RAB11B (TA346586) were from Origene, Rockville, MD, USA.

Techniques: Confocal Microscopy, Incubation, Microscopy, Western Blot, Control