Les normes, comment?

La description normalisée des ressources d’enseignement et d’apprentissage requiert l’utilisation d’outils d’implantation conformes aux conventions d’un standard ou d’une norme internationale et un réseau d’entraide et d’accompagnement

Direct binding of vinculin and Rab5.

<p>(A) Endogenous vinculin and Rab5 were immunoprecipitated from Cos-7 lysates with anti-Rab5 or anti-vinculin antibodies. Vinculin and Rab5 were assayed by western blotting using specific antibodies to vinculin and Rab5. (B) HA-Rab5 (S34N), HA-Rab5 (Q79L) and HA-Rab5 (WT) were transiently expressed in Cos-7 cells and subjected to immunoprecipitation with anti-HA antibody. Note that immunoprecipitation of HA-Rab5 (WT) was carried out with GTPγS and GDP. Proteins were assayed by western blotting. The graph shows mean ± S.E. values of three independent experiments (C) A pull-down assay from a Cos-7 lysate was performed using GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L). Vinculin bound to the beads was assayed by western blotting, and GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) on a PVDF membrane were stained with Ponceau S. The graph shows mean ± S.E. values of three independent experiments (D) GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) were incubated with purified His-vinculin, and a pull-down assay was performed using glutathione beads. His-Vinculin bound to the beads was assayed by western blotting, and GST, GST-Rab5 (S34N), and GST-Rab5 (Q79L) on a PVDF membrane were stained with Ponceau S. The graph shows mean ± S.E. values of three independent experiments.</p

Determination of the Rab5-binding domain of vinculin.

<p>(A) Schematic of vinculin deletion mutants. (B) GST-Rab5 (Q79L) was incubated with purified His-vinculin deletion mutants, and a His-pull-down assay was performed. The beads were assayed by western blotting. (C) GFP, GFP-Vin1-258, GFP-Vin1-880, GFP-Vin258-880, GFP-Vin881-1066 and GFP-vinculin (full length) were coexpressed with HA-Rab5 in Cos-7 cells and immunostained with anti-HA antibody. (D) pHrodo red-labeled <i>S. aureus</i> was added to the medium of Cos-7 cells expressing each vinculin deletion mutant and incubated for 2 h at 37°C. The graph shows mean ± S.E. values of six independent experiments, **<i>p</i><0.01. (E) Cos-7 cells were transfected with HA-Rab5 (WT) and vinculin deletion mutants. <i>S. aureus</i> was added to the medium of transfected cells and incubated for 60 min at 37°C. The cells were lysed and subjected to a GST-R5BD pull-down assay. GST-R5BD-bound beads and lysates were assayed by western blotting with anti-HA antibody.</p

Effects of <i>S. aureus</i> on vinculin-Rab5 binding and Rab5-GTP.

<p>(A) S. aureus was added to the medium of HA-Rab5 (WT)-transfected Cos-7 cells and incubated with <i>S. aureus</i> for the indicated time at 37°C. The cells were lysed and subjected to immunoprecipitation with anti-HA antibody. Immunocomplexes were assayed by western blotting with anti-HA and anti-vinculin antibodies. Proteins levels were quantified using ImageJ in three independent experiments. (B) <i>S. aureus</i> was added to the medium of Cos-7 cells expressing GFP-Rab5 (WT) and RFP-vinculin (WT) and incubated for the indicated time at 37°C. The cells were fixed and immunostained with <i>S. aureus</i> antibodies. (C) HeLa cells were transfected with GFP-Rab5 (WT) and vinculin siRNA or control siRNA. <i>S. aureus</i> was added to the medium of transfected cell and incubated for the indicated time at 37°C. The cells were lysed and subjected to a GST-R5BD pull-down assay. GST-R5BD-bound beads and lysates were assayed by western blotting with anti-GFP antibody. Rab5-GTP levels were normalized to total GFP-Rab5 levels and quantified using ImageJ.</p

Effects of vinculin and Rab5 knockdown on <i>S. aureus</i> infection in murine lung.

<p>(A and B) Mouse lungs were transfected with vinculin and Rab5 siRNA. After 3 days, lungs were homogenized and treated with anti-vinculin, anti-Rab5, and anti-GAPDH (internal control) antibodies. (C and D) Murine lungs in which vinculin or Rab5 had been knocked down were infected with <i>S</i>. <i>aureus</i>. Lung tissues were analyzed for colony formation. The graph shows the mean ± S.E. of five independent experiments. (E–F) Murine lung in which vinculin (E and G) or Rab5 (F and H) had been knocked down was infected with <i>S</i>. <i>aureus</i>. The murine lung homogenates were assayed with anti-JNK, anti-phosphor-JNK, anti-ERK, anti-phosphor-Erk, anti-p38, anti-phosphor-p38, anti-IL-6, and anti-GAPDH (internal control) antibodies by western blotting.</p

Effect of vinculin and Rab5 knockdown on <i>S. aureus</i>-induced IL-6 expression.

<p>(A and B) <i>S. aureus</i> was added to the medium of HeLa cells with vinculin (A) or Rab5 (B) knockdown and incubated for 60 min at 37°C. The cell lysates were assayed with anti-JNK, anti-phosphor-JNK, anti-ERK, anti-phosphor-Erk, anti-p38, and anti-phosphor-p38 antibodies by western blotting. (C and D) <i>S. aureus</i> was added to the medium of HeLa cells with vinculin or Rab5 knockdown and incubated for 16 h. The cell lysate (C) and conditioned medium (D) were assayed with anti-IL-6 antibody by western blotting. GAPDH and albumin were internal controls.</p

Effects of vinculin and Rab5 knockdown on cellular uptake.

<p>(A) siRNAs for vinculin were transfected into HeLa cells, and cell lysates were assayed by western blotting using anti-vinculin, anti-Rab5, and anti-GAPDH antibodies (internal control). (B–F) Various uptake indicators were added to the media of vinculin knockdown cells and incubated at 37°C for 2 h. Uptake of pHrodo red-labeled <i>S. aureus</i> decreased, but that of transferrin Alexa 555, albumin Alexa 555, Lucifer yellow, and FM 4-64 did not decrease. Error bar: n = 3−6±SE, **<i>p</i><0.01. (G) siRNAs for Rab5 were transfected into HeLa cells and cell lysates were assayed by western blotting with anti-Rab5, anti-vinculin, and anti-GAPDH antibodies. (H–L) HeLa cells transfected with Rab5 siRNA. Various uptake indicators were added to the media of Rab5 knockdown cells and incubated at 37°C for 2 h. Uptake of pHrodo red-labeled <i>S. aureus</i>, transferrin Alexa 555, albumin Alexa 555, Lucifer yellow, and FM 4-64 was decreased. Error bar: n = 3−6±SE, **<i>p</i><0.01.</p