rabbit anti arf6  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit anti arf6
    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged <t>Arf6</t> T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " width="250" height="auto" />
    Rabbit Anti Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti arf6/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti arf6 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling"

    Article Title: Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021708

    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " title="... with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels.

    Techniques Used: Stable Transfection, Transfection, Incubation, Confocal Microscopy, Staining, Expressing, Western Blot

    ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. " title="... with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings.

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot, Incubation, Staining, Confocal Microscopy

    rabbit anti arf6  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit anti arf6
    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged <t>Arf6</t> T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " width="250" height="auto" />
    Rabbit Anti Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti arf6/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti arf6 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling"

    Article Title: Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021708

    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " title="... with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels.

    Techniques Used: Stable Transfection, Transfection, Incubation, Confocal Microscopy, Staining, Expressing, Western Blot

    ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. " title="... with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings.

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot, Incubation, Staining, Confocal Microscopy

    arf 6  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc arf 6
    The nBSA enters the cells through <t>Arf-6</t> dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.
    Arf 6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/arf 6/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    arf 6 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy"

    Article Title: Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    Journal: Theranostics

    doi: 10.7150/thno.16587

    The nBSA enters the cells through Arf-6 dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.
    Figure Legend Snippet: The nBSA enters the cells through Arf-6 dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.

    Techniques Used: Labeling, Transfection

    arf 6  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc arf 6
    The LA-nBSA enters the cells through <t>Arf-6-dependent</t> endocytosis. (A,B) Confocal images of MCF-7 cells treated with 1 mg mL –1 FITC-labeled nBSA and FITC-labeled LA-nBSA for 20 h. Arf-6 and EEA1 were detected with primary antibody against Arf-6 and EEA1, respectively. (C) DsRed-Rab7 transfected MCF-7 cells and then treated with 1 mg mL −1 FITC-labeled nBSA and LA-nBSA for 20 h, respectively. (D) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA and LA-nBSA for 20 h, respectively, and then co-treated with Lyso-Tracker Red probes for 1 h. Scale bars: 10 μm.
    Arf 6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    arf 6 - by Bioz Stars, 2023-01
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    Images

    1) Product Images from "The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery"

    Article Title: The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery

    Journal: Drug Delivery

    doi: 10.1080/10717544.2018.1461954

    The LA-nBSA enters the cells through Arf-6-dependent endocytosis. (A,B) Confocal images of MCF-7 cells treated with 1 mg mL –1 FITC-labeled nBSA and FITC-labeled LA-nBSA for 20 h. Arf-6 and EEA1 were detected with primary antibody against Arf-6 and EEA1, respectively. (C) DsRed-Rab7 transfected MCF-7 cells and then treated with 1 mg mL −1 FITC-labeled nBSA and LA-nBSA for 20 h, respectively. (D) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA and LA-nBSA for 20 h, respectively, and then co-treated with Lyso-Tracker Red probes for 1 h. Scale bars: 10 μm.
    Figure Legend Snippet: The LA-nBSA enters the cells through Arf-6-dependent endocytosis. (A,B) Confocal images of MCF-7 cells treated with 1 mg mL –1 FITC-labeled nBSA and FITC-labeled LA-nBSA for 20 h. Arf-6 and EEA1 were detected with primary antibody against Arf-6 and EEA1, respectively. (C) DsRed-Rab7 transfected MCF-7 cells and then treated with 1 mg mL −1 FITC-labeled nBSA and LA-nBSA for 20 h, respectively. (D) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA and LA-nBSA for 20 h, respectively, and then co-treated with Lyso-Tracker Red probes for 1 h. Scale bars: 10 μm.

    Techniques Used: Labeling, Transfection

    anti arf6  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti arf6
    Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or <t>Arf6‐GFP</t> and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.
    Anti Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti arf6/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti arf6 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication"

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    Journal: The EMBO Journal

    doi: 10.15252/embj.2021107664

    Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or Arf6‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.
    Figure Legend Snippet: Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or Arf6‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.

    Techniques Used: Transfection, Expressing, Fluorescence, Staining, Software, MANN-WHITNEY, Western Blot, Labeling

    A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).
    Figure Legend Snippet: A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

    Techniques Used: Transfection, Incubation, Binding Assay, Staining, Marker

    A Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry‐BspF (grayscale panels), incubated on ice with AlexaFluor™488‐Cholera Toxin subunit B (CTxB; green) and shifted to 37°C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). B Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry‐BspF, or HA‐BspF over a 30‐min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a two‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, and Rab6a/a′ depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA‐mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a), or Rab6a/a′ (siRab6a/a′) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with siNT control cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). E Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock‐infected or infected with wild‐type (2308), Δ bspF , complemented ∆ bspF (Δ bspF::bspF ), ∆ bspB or complemented ∆ bspB (Δ bspB::bspB ) bacteria for 24 h, incubated for 30 min with AlexaFluor™488‐CTxB on ice for binding followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with mock‐infected cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). F rBCV biogenesis in BMMs treated with either non‐targeting siNT, siRab6a/a′, or siRab8a siRNAs and infected with wild‐type (2308) bacteria. Data are means ± SD of n = 3 independent experiments, in which 100 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) compared with control (2308). G–I Brucella replication in BMMs treated with non‐targeting siRNAs (siNT), or siRNAs against Rab6a/a′ (siRab6a/a′) (G), Rab8a (siRab8a) (H), or Arf6 (siArf6) (I) and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.
    Figure Legend Snippet: A Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry‐BspF (grayscale panels), incubated on ice with AlexaFluor™488‐Cholera Toxin subunit B (CTxB; green) and shifted to 37°C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). B Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry‐BspF, or HA‐BspF over a 30‐min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a two‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, and Rab6a/a′ depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA‐mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a), or Rab6a/a′ (siRab6a/a′) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with siNT control cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). E Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock‐infected or infected with wild‐type (2308), Δ bspF , complemented ∆ bspF (Δ bspF::bspF ), ∆ bspB or complemented ∆ bspB (Δ bspB::bspB ) bacteria for 24 h, incubated for 30 min with AlexaFluor™488‐CTxB on ice for binding followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with mock‐infected cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). F rBCV biogenesis in BMMs treated with either non‐targeting siNT, siRab6a/a′, or siRab8a siRNAs and infected with wild‐type (2308) bacteria. Data are means ± SD of n = 3 independent experiments, in which 100 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) compared with control (2308). G–I Brucella replication in BMMs treated with non‐targeting siRNAs (siNT), or siRNAs against Rab6a/a′ (siRab6a/a′) (G), Rab8a (siRab8a) (H), or Arf6 (siArf6) (I) and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Techniques Used: Fluorescence, Transfection, Incubation, Staining, Marker, Western Blot, Binding Assay, Infection

    Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.
    Figure Legend Snippet: Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Techniques Used: Transfection, Western Blot, Immunoprecipitation, Magnetic Beads, SDS Page, Activity Assay, Infection

    Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.
    Figure Legend Snippet: Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

    Techniques Used: Fluorescence, Transfection, Labeling

    A Representative confocal micrographs of BMMs infected with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF::bspF ) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6‐positive vesicles and rBCVs. B Recruitment of Stx6‐positive vesicles to rBCVs (expressed as percentage of Stx6‐positive BCVs) in BMMs infected for 24 h with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF :: bspF ) bacteria. Data are means ± SD from n = 3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared with 2308‐infected BMMs as determined by one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, Rab6a/a′, and Stx6 depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D–F Recruitment of Stx6‐positive vesicles to rBCVs in BMMs treated with non‐targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E), or Rab6a/a′ (siRab6a/a′) (F) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions; ns, not significant. G Quantification of CTxB retrograde transport in BMMs following siRNA‐mediated depletion of Stx6 (siStx6) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference ( P = 0.0114, unpaired Student’s t ‐test) compared with the siNT control. H Brucella replication in BMMs treated with either non‐targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. I Model of BspF remodeling of TGN‐RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE‐TGN transport, which alters TGN‐derived vesicular traffic and redirects Stx6‐positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth. Source data are available online for this figure.
    Figure Legend Snippet: A Representative confocal micrographs of BMMs infected with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF::bspF ) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6‐positive vesicles and rBCVs. B Recruitment of Stx6‐positive vesicles to rBCVs (expressed as percentage of Stx6‐positive BCVs) in BMMs infected for 24 h with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF :: bspF ) bacteria. Data are means ± SD from n = 3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared with 2308‐infected BMMs as determined by one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, Rab6a/a′, and Stx6 depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D–F Recruitment of Stx6‐positive vesicles to rBCVs in BMMs treated with non‐targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E), or Rab6a/a′ (siRab6a/a′) (F) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions; ns, not significant. G Quantification of CTxB retrograde transport in BMMs following siRNA‐mediated depletion of Stx6 (siStx6) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference ( P = 0.0114, unpaired Student’s t ‐test) compared with the siNT control. H Brucella replication in BMMs treated with either non‐targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. I Model of BspF remodeling of TGN‐RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE‐TGN transport, which alters TGN‐derived vesicular traffic and redirects Stx6‐positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth. Source data are available online for this figure.

    Techniques Used: Infection, Marker, Western Blot, Binding Assay, Incubation, Inhibition, Derivative Assay


    Figure Legend Snippet:

    Techniques Used: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

    arf6  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arf6
    Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti arf6 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti arf6 antibody
    Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of <t>Arf6</t> activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.
    Rabbit Anti Arf6 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus"

    Article Title: ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus

    Journal: eNeuro

    doi: 10.1523/ENEURO.0111-20.2020

    Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of Arf6 activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.
    Figure Legend Snippet: Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of Arf6 activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.

    Techniques Used: Mutagenesis, Selection, Expressing, Modification, Functional Assay, Immunohistochemistry, Western Blot, Two Tailed Test, Activation Assay, Pull Down Assay

    arf6  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arf6
    DPBA-induced EGFR endocytosis is clathrin-independent but lipid raft-mediated. a EGF-induced EGFR endocytosis was impaired by inhibition of EGFR kinase activity or clathrin. A549 was treated with EGF (20 ng/ml) in the presence or absence of gefitinib (10 μM), cetuximab (5 μg/ml), pitstop2 (5 μM), or clathrin siRNA for 30 min. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). b DPBA-induced EGFR endocytosis was blocked by a lipid raft inhibitor MCD. A549 and H1975 were treated with DPBA (6 μM) in the presence or absence of afatinib (10 μM), AZD9291 (10 μM), pitstop2 (5 μM), MCD (1 mg/ml), or clathrin siRNA for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). c DPBA degraded both EGFR WT and EGFR KD. HEK-293T transfected with pCMV-HA, pCMV-HA EGFR WT, or pCMV-HA EGFR KD was treated with EGF (20 ng/ml) for 30 min or DPBA (6 μM) for 24 h. Exogenous EGFR was subjected to pull-down with anti-HA antibody. EGFR and p-EGFR (Y1068) levels were measured by Western blot. d A549 and H1975 were treated with indicated concentrations of DPBA in the presence or absence of MCD (1 mg/ml) for 24 h. Cell viability was measured by MTT assay, ** P < 0.01, *** P < 0.001 vs. DPBA, n = 3. e A549 and H1975 were treated with DPBA (6 μM) with or without MCD (1 mg/ml) for 24 h. Total EGFR, p-EGFR (Y1068), Akt, p-Akt (T308), ERK, p-ERK (T202/Y204), STAT3, p-STAT3 (S727), and PARP expression levels were measured by Western blot. f DPBA induced EGFR accumulation in lipid rafts. A549 was treated with DPBA (6 μM) for 3 h. EGFR distributions in lipid rafts were detected by density gradient centrifugation and Western blot. TfR was a non-lipid raft marker while caveolin-1 and flotillin-1 were lipid raft markers. g Flotillin-1 knockdown blocked DPBA-induced EGFR endocytosis. A549 was transfected with dynamin 2 siRNA, flotillin-1 siRNA, caveolin-1 siRNA, GRAF1 siRNA, RhoA siRNA, and <t>Arf6</t> siRNA for 48 h, followed by DPBA treatment (6 μM) for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). h Flotillin-1 knockdown inhibited DPBA-induced EGFR degradation. A549 was transfected with flotillin-1 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot. i DPBA enhanced interaction of EGFR and flotillin-1. A549 was treated with DPBA (6 μM) for 3, 6, and 12 h. Interactions between EGFR and flotillin-1 were detected by co-IP. j A549 was transfected with dynamin 2 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot
    Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth"

    Article Title: Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00251-2

    DPBA-induced EGFR endocytosis is clathrin-independent but lipid raft-mediated. a EGF-induced EGFR endocytosis was impaired by inhibition of EGFR kinase activity or clathrin. A549 was treated with EGF (20 ng/ml) in the presence or absence of gefitinib (10 μM), cetuximab (5 μg/ml), pitstop2 (5 μM), or clathrin siRNA for 30 min. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). b DPBA-induced EGFR endocytosis was blocked by a lipid raft inhibitor MCD. A549 and H1975 were treated with DPBA (6 μM) in the presence or absence of afatinib (10 μM), AZD9291 (10 μM), pitstop2 (5 μM), MCD (1 mg/ml), or clathrin siRNA for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). c DPBA degraded both EGFR WT and EGFR KD. HEK-293T transfected with pCMV-HA, pCMV-HA EGFR WT, or pCMV-HA EGFR KD was treated with EGF (20 ng/ml) for 30 min or DPBA (6 μM) for 24 h. Exogenous EGFR was subjected to pull-down with anti-HA antibody. EGFR and p-EGFR (Y1068) levels were measured by Western blot. d A549 and H1975 were treated with indicated concentrations of DPBA in the presence or absence of MCD (1 mg/ml) for 24 h. Cell viability was measured by MTT assay, ** P < 0.01, *** P < 0.001 vs. DPBA, n = 3. e A549 and H1975 were treated with DPBA (6 μM) with or without MCD (1 mg/ml) for 24 h. Total EGFR, p-EGFR (Y1068), Akt, p-Akt (T308), ERK, p-ERK (T202/Y204), STAT3, p-STAT3 (S727), and PARP expression levels were measured by Western blot. f DPBA induced EGFR accumulation in lipid rafts. A549 was treated with DPBA (6 μM) for 3 h. EGFR distributions in lipid rafts were detected by density gradient centrifugation and Western blot. TfR was a non-lipid raft marker while caveolin-1 and flotillin-1 were lipid raft markers. g Flotillin-1 knockdown blocked DPBA-induced EGFR endocytosis. A549 was transfected with dynamin 2 siRNA, flotillin-1 siRNA, caveolin-1 siRNA, GRAF1 siRNA, RhoA siRNA, and Arf6 siRNA for 48 h, followed by DPBA treatment (6 μM) for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). h Flotillin-1 knockdown inhibited DPBA-induced EGFR degradation. A549 was transfected with flotillin-1 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot. i DPBA enhanced interaction of EGFR and flotillin-1. A549 was treated with DPBA (6 μM) for 3, 6, and 12 h. Interactions between EGFR and flotillin-1 were detected by co-IP. j A549 was transfected with dynamin 2 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot
    Figure Legend Snippet: DPBA-induced EGFR endocytosis is clathrin-independent but lipid raft-mediated. a EGF-induced EGFR endocytosis was impaired by inhibition of EGFR kinase activity or clathrin. A549 was treated with EGF (20 ng/ml) in the presence or absence of gefitinib (10 μM), cetuximab (5 μg/ml), pitstop2 (5 μM), or clathrin siRNA for 30 min. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). b DPBA-induced EGFR endocytosis was blocked by a lipid raft inhibitor MCD. A549 and H1975 were treated with DPBA (6 μM) in the presence or absence of afatinib (10 μM), AZD9291 (10 μM), pitstop2 (5 μM), MCD (1 mg/ml), or clathrin siRNA for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). c DPBA degraded both EGFR WT and EGFR KD. HEK-293T transfected with pCMV-HA, pCMV-HA EGFR WT, or pCMV-HA EGFR KD was treated with EGF (20 ng/ml) for 30 min or DPBA (6 μM) for 24 h. Exogenous EGFR was subjected to pull-down with anti-HA antibody. EGFR and p-EGFR (Y1068) levels were measured by Western blot. d A549 and H1975 were treated with indicated concentrations of DPBA in the presence or absence of MCD (1 mg/ml) for 24 h. Cell viability was measured by MTT assay, ** P < 0.01, *** P < 0.001 vs. DPBA, n = 3. e A549 and H1975 were treated with DPBA (6 μM) with or without MCD (1 mg/ml) for 24 h. Total EGFR, p-EGFR (Y1068), Akt, p-Akt (T308), ERK, p-ERK (T202/Y204), STAT3, p-STAT3 (S727), and PARP expression levels were measured by Western blot. f DPBA induced EGFR accumulation in lipid rafts. A549 was treated with DPBA (6 μM) for 3 h. EGFR distributions in lipid rafts were detected by density gradient centrifugation and Western blot. TfR was a non-lipid raft marker while caveolin-1 and flotillin-1 were lipid raft markers. g Flotillin-1 knockdown blocked DPBA-induced EGFR endocytosis. A549 was transfected with dynamin 2 siRNA, flotillin-1 siRNA, caveolin-1 siRNA, GRAF1 siRNA, RhoA siRNA, and Arf6 siRNA for 48 h, followed by DPBA treatment (6 μM) for 6 h. EGFR endocytosis was observed by immunofluorescence (magnification, ×630; scale bar, 10 μm). h Flotillin-1 knockdown inhibited DPBA-induced EGFR degradation. A549 was transfected with flotillin-1 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot. i DPBA enhanced interaction of EGFR and flotillin-1. A549 was treated with DPBA (6 μM) for 3, 6, and 12 h. Interactions between EGFR and flotillin-1 were detected by co-IP. j A549 was transfected with dynamin 2 siRNA (100 nM) for 48 h, followed by DPBA treatment (6 μM) for 24 h. EGFR degradation was detected by Western blot

    Techniques Used: Inhibition, Activity Assay, Immunofluorescence, Transfection, Western Blot, MTT Assay, Expressing, Gradient Centrifugation, Marker, Co-Immunoprecipitation Assay

    polyclonal rabbit anti arf6  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal rabbit anti arf6
    Polyclonal Rabbit Anti Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal rabbit anti arf6  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal rabbit anti arf6
    Polyclonal Rabbit Anti Arf6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti arf6 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arf6 antibody
    Endothelial cell <t>Arf6</t> deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.
    Anti Arf6 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection"

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00303-20

    Endothelial cell Arf6 deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.
    Figure Legend Snippet: Endothelial cell Arf6 deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.

    Techniques Used: Permeability, Staining

    Pharmacologic inhibition of ARF6 increases survival rates and reduces disease severity due to MDR GNB infection. ARF6 inhibitors (A6-5093, NAV-2729, and A6-4424) increase survival (n = 20 mice for all groups, except those for A6-5093 [10 mice] and colistin [19 mice]) (A) and reduce lung bacterial burden (B) of immunosuppressed mice (200 mg/kg i.p. cyclophosphamide and 250 mg/kg subcutaneous cortisone acetate given on days −2 and +3 relative to infection) with MDR A. baumannii pneumonia. Lungs were harvested on day +4 relative to infection. *, P < 0.05 versus vehicle-treated mice; **, P < 0.02 versus all others in panel A; #, P < 0.05 versus vehicle-treated mice; ***, P < 0.03 versus all others in panel B. (C and D) ARF6 inhibitors increase survival rate of immunosuppressed mice infected with MDR CPKP (n = 10/group) (C) or MDR P. aeruginosa (n = 20/group) (D). Log rank test for all analyses of survival and Wilcoxon rank sum test for lung bacterial burden.
    Figure Legend Snippet: Pharmacologic inhibition of ARF6 increases survival rates and reduces disease severity due to MDR GNB infection. ARF6 inhibitors (A6-5093, NAV-2729, and A6-4424) increase survival (n = 20 mice for all groups, except those for A6-5093 [10 mice] and colistin [19 mice]) (A) and reduce lung bacterial burden (B) of immunosuppressed mice (200 mg/kg i.p. cyclophosphamide and 250 mg/kg subcutaneous cortisone acetate given on days −2 and +3 relative to infection) with MDR A. baumannii pneumonia. Lungs were harvested on day +4 relative to infection. *, P < 0.05 versus vehicle-treated mice; **, P < 0.02 versus all others in panel A; #, P < 0.05 versus vehicle-treated mice; ***, P < 0.03 versus all others in panel B. (C and D) ARF6 inhibitors increase survival rate of immunosuppressed mice infected with MDR CPKP (n = 10/group) (C) or MDR P. aeruginosa (n = 20/group) (D). Log rank test for all analyses of survival and Wilcoxon rank sum test for lung bacterial burden.

    Techniques Used: Inhibition, Infection

    ARF6 inhibition reduces organ permeability and disease severity without affecting the inflammatory response to A. baumannii pneumonia. (A) ARF6 inhibitors (NAV-2729 and A6-5093) decreased permeability in the lungs and kidneys of A. baumannii HUMC1-infected mice. ‡, P < 0.05 versus other treatments. (B) NAV-2729 (30 mg/kg) reduced severity of A. baumannii infection, as shown by histopathological examination of lungs with H&E stain and spleen with Gram stain. An asterisk denotes necrobiosis in spleen harvested from vehicle-treated mice and, to a lesser extent, colistin-treated mice, but not from mice treated with NAV-2729. Bars represent 100 μM. (C) IL-6 levels in lungs and plasma of A. baumannii HUMC1-infected mice and treated with vehicle control, NAV-2729, or colistin. §, P < 0.02 versus uninfected mice. Organs of neutropenic mice were harvested on day +3 (A) and day +4 (B and C) relative to infection. Data in panels A and C are presented as the medians ± interquartile ranges.
    Figure Legend Snippet: ARF6 inhibition reduces organ permeability and disease severity without affecting the inflammatory response to A. baumannii pneumonia. (A) ARF6 inhibitors (NAV-2729 and A6-5093) decreased permeability in the lungs and kidneys of A. baumannii HUMC1-infected mice. ‡, P < 0.05 versus other treatments. (B) NAV-2729 (30 mg/kg) reduced severity of A. baumannii infection, as shown by histopathological examination of lungs with H&E stain and spleen with Gram stain. An asterisk denotes necrobiosis in spleen harvested from vehicle-treated mice and, to a lesser extent, colistin-treated mice, but not from mice treated with NAV-2729. Bars represent 100 μM. (C) IL-6 levels in lungs and plasma of A. baumannii HUMC1-infected mice and treated with vehicle control, NAV-2729, or colistin. §, P < 0.02 versus uninfected mice. Organs of neutropenic mice were harvested on day +3 (A) and day +4 (B and C) relative to infection. Data in panels A and C are presented as the medians ± interquartile ranges.

    Techniques Used: Inhibition, Permeability, Infection, Staining

    A. baumannii-mediated HUVEC permeability is induced by LPS-TLR4 signaling through ARF6. (A) A. baumannii HUMC1 (virulent MDR) cells and supernatants (sup) induce HUVEC permeability to a level similar to that of E. coli LPS. Removing LPS by polymyxin B blocks this induction. ATCC 17978 (avirulent, drug-sensitive) cells and supernatants do not induce permeability. *, P < 0.005 versus HUMC1, HUMC1 supernatant, or E. coli LPS. (B) A permeability assay was conducted with HUMC1 or its supernatant in the presence of 50 μg/ml anti-TLR4 or isotype-matched control antibodies (Ab). *, P < 0.001 versus isotype-matched antibody. (C) ARF6-GTP pulldown assays show that A. baumannii HUMC1 (AB) induces ARF6 activation and NAV-2729 blocks it. Quantification of the ARF6-GTP/total ARF6 ratio by densitometer (3 independent experiments) shows A. baumannii induces endothelial cell ARF6 activation 2-fold, while NAV-2729 inhibits this activation. *, P < 0.05 versus all other comparators. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) A HUVEC permeability assay was conducted with HUMC1 in the presence of 50 μM ARF6 inhibitor (NAV-2729 or A6-4424). *, P < 0.0015 versus HUMC1 cells.
    Figure Legend Snippet: A. baumannii-mediated HUVEC permeability is induced by LPS-TLR4 signaling through ARF6. (A) A. baumannii HUMC1 (virulent MDR) cells and supernatants (sup) induce HUVEC permeability to a level similar to that of E. coli LPS. Removing LPS by polymyxin B blocks this induction. ATCC 17978 (avirulent, drug-sensitive) cells and supernatants do not induce permeability. *, P < 0.005 versus HUMC1, HUMC1 supernatant, or E. coli LPS. (B) A permeability assay was conducted with HUMC1 or its supernatant in the presence of 50 μg/ml anti-TLR4 or isotype-matched control antibodies (Ab). *, P < 0.001 versus isotype-matched antibody. (C) ARF6-GTP pulldown assays show that A. baumannii HUMC1 (AB) induces ARF6 activation and NAV-2729 blocks it. Quantification of the ARF6-GTP/total ARF6 ratio by densitometer (3 independent experiments) shows A. baumannii induces endothelial cell ARF6 activation 2-fold, while NAV-2729 inhibits this activation. *, P < 0.05 versus all other comparators. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) A HUVEC permeability assay was conducted with HUMC1 in the presence of 50 μM ARF6 inhibitor (NAV-2729 or A6-4424). *, P < 0.0015 versus HUMC1 cells.

    Techniques Used: Permeability, Activation Assay

    A. baumannii-mediated HUVEC permeability is induced through the MyD88/ARNO/ARF6 pathway. (A) Successful downregulation of gene expression (quantitative reverse transcription-PCR [qRT-PCR]) using siRNA constructs targeting ARF6, ARNO, or MyD88 in HUVECs. *, P < 0.001 versus untransfected HUVECs (2 experiments). (B and C) ARF6 expression as determined by qRT-PCR and Western blotting (B) and A. baumannii-mediated permeability using FITC-dextran (C) of HUVECs transfected with siRNA constructs targeting MyD88, ARNO, ARF6, or scrambled sequence (control). *, P < 0.008 versus control siRNA plus A. baumannii (B) and P < 0.002 versus control siRNA plus A. baumannii (C).
    Figure Legend Snippet: A. baumannii-mediated HUVEC permeability is induced through the MyD88/ARNO/ARF6 pathway. (A) Successful downregulation of gene expression (quantitative reverse transcription-PCR [qRT-PCR]) using siRNA constructs targeting ARF6, ARNO, or MyD88 in HUVECs. *, P < 0.001 versus untransfected HUVECs (2 experiments). (B and C) ARF6 expression as determined by qRT-PCR and Western blotting (B) and A. baumannii-mediated permeability using FITC-dextran (C) of HUVECs transfected with siRNA constructs targeting MyD88, ARNO, ARF6, or scrambled sequence (control). *, P < 0.008 versus control siRNA plus A. baumannii (B) and P < 0.002 versus control siRNA plus A. baumannii (C).

    Techniques Used: Permeability, Expressing, Quantitative RT-PCR, Construct, Western Blot, Transfection, Sequencing

    Model of GNB-induced vascular leak and the role of ARF6 inhibitor in preserving vascular integrity and increasing survival in bacterial infections. (A) In a normal host, the vasculature is held intact by VE-cadherin localized to the cell-cell junctions. (B) In bacterial sepsis (e.g., A. baumannii [AB]), LPS-induced endotoxemia triggers a robust host inflammatory response by TLR4-mediated activation of MyD88/NF-κB required for clearing the infection. (C) Bacterial LPS also triggers ARF6 activation via MyD88/ARNO, which leads to intercellular recruitment of VE-cadherin, resulting in increased vascular leak, tissue edema, organ failure, and, ultimately, death. (D) ARF6 inhibitors prevent ARF6 activation, which reduces VE-cadherin internalization, resulting in the preservation of vascular integrity without affecting the inflammatory immune response.
    Figure Legend Snippet: Model of GNB-induced vascular leak and the role of ARF6 inhibitor in preserving vascular integrity and increasing survival in bacterial infections. (A) In a normal host, the vasculature is held intact by VE-cadherin localized to the cell-cell junctions. (B) In bacterial sepsis (e.g., A. baumannii [AB]), LPS-induced endotoxemia triggers a robust host inflammatory response by TLR4-mediated activation of MyD88/NF-κB required for clearing the infection. (C) Bacterial LPS also triggers ARF6 activation via MyD88/ARNO, which leads to intercellular recruitment of VE-cadherin, resulting in increased vascular leak, tissue edema, organ failure, and, ultimately, death. (D) ARF6 inhibitors prevent ARF6 activation, which reduces VE-cadherin internalization, resulting in the preservation of vascular integrity without affecting the inflammatory immune response.

    Techniques Used: Preserving, Activation Assay, Infection

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    Cell Signaling Technology Inc rabbit anti arf6
    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged <t>Arf6</t> T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " width="250" height="auto" />
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    The nBSA enters the cells through <t>Arf-6</t> dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.
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    Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or <t>Arf6‐GFP</t> and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.
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    Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or <t>Arf6‐GFP</t> and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.
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    Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of <t>Arf6</t> activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.
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    Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of <t>Arf6</t> activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.
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    Endothelial cell <t>Arf6</t> deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.
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    ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels. " width="100%" height="100%">

    Journal: PLoS ONE

    Article Title: Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

    doi: 10.1371/journal.pone.0021708

    Figure Lengend Snippet: ( A ) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. ( B ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( C ) or Cdc42 ( E ) or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 ( D ) or Cdc42 ( F ) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml 125 I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound 125 I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels.

    Article Snippet: The following primary antibodies were used: mouse anti-HA.11 (Covance, Nordic Biosite, Täby, Sweden); mouse anti-early endosomal antigen (EEA) 1, mouse anti-Hsp90, mouse anti-flotillin 1, mouse anti-flotillin 2 (BD Biosciences Transduction Laboratories, Lexington, KY); mouse anti-CHC (RDI division of Fitzgerald Industries, Concorde, MA); goat anti-CHC, rabbit anti-FGFR1, rabbit anti-FGFR3 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-HA, mouse anti-Myc Tag, clone 4A6 (Millipore, Billerica, MA); mouse anti-phospho-FGFR, rabbit anti-phospho-FRS2α (Y196), rabbit anti-MAPK (p42/p44), mouse anti-phospho-MAPK (p42/p44), rabbit anti-Cdc42, rabbit anti-Arf6 (Cell Signaling Technology, Danvers, MA); rabbit anti-flotillin-2, mouse anti-γ-tubulin (Sigma-Aldrich, St.Louis, MO).

    Techniques: Stable Transfection, Transfection, Incubation, Confocal Microscopy, Staining, Expressing, Western Blot

    ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in <xref ref-type= Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. " width="100%" height="100%">

    Journal: PLoS ONE

    Article Title: Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

    doi: 10.1371/journal.pone.0021708

    Figure Lengend Snippet: ( A ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in Materials and Methods . The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. ( B ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. ( C ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. ( D ) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( E ) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. ( F ) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings.

    Article Snippet: The following primary antibodies were used: mouse anti-HA.11 (Covance, Nordic Biosite, Täby, Sweden); mouse anti-early endosomal antigen (EEA) 1, mouse anti-Hsp90, mouse anti-flotillin 1, mouse anti-flotillin 2 (BD Biosciences Transduction Laboratories, Lexington, KY); mouse anti-CHC (RDI division of Fitzgerald Industries, Concorde, MA); goat anti-CHC, rabbit anti-FGFR1, rabbit anti-FGFR3 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-HA, mouse anti-Myc Tag, clone 4A6 (Millipore, Billerica, MA); mouse anti-phospho-FGFR, rabbit anti-phospho-FRS2α (Y196), rabbit anti-MAPK (p42/p44), mouse anti-phospho-MAPK (p42/p44), rabbit anti-Cdc42, rabbit anti-Arf6 (Cell Signaling Technology, Danvers, MA); rabbit anti-flotillin-2, mouse anti-γ-tubulin (Sigma-Aldrich, St.Louis, MO).

    Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Incubation, Staining, Confocal Microscopy

    The nBSA enters the cells through Arf-6 dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.

    Journal: Theranostics

    Article Title: Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    doi: 10.7150/thno.16587

    Figure Lengend Snippet: The nBSA enters the cells through Arf-6 dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.

    Article Snippet: Antibodies against LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, Clathrin, Caveolin were from Cell Signaling Technology.

    Techniques: Labeling, Transfection

    Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or Arf6‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: Quantification of ss‐eGFP‐FKBP F36 M trafficking in HeLa(M)‐C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry‐BspF (mCherry‐BspF). Rapamycin was added to initiate secretory traffic of ss‐eGFP‐FKBP F36 M and its colocalization with Calnexin (ER), ERGIC‐53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) were scored over a 60‐min time course. Data are means ± SD from n = 3 independent experiments. Asterisks indicate statistically significant differences between mCherry‐ and mCherry‐BspF‐expressing cells as determined by a two‐way ANOVA with Sidak’s multiple comparisons test ( P < 0.05). Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce GFP‐TGN38 and mCherry‐BspF and stained for F‐actin with AlexaFluor™647‐phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets). Quantification of colocalization between mCherry‐BspF and GFP‐TGN38 in untreated (−CytoD) and Cytochalasin D‐treated (+CytoD) HeLa cells. Regions of interests (ROI, representative shown as insets in panel B) were randomly selected, and a Pearson’s correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug‐in. Data are means ± SD from n = 3 independent experiments in which 2 ROIs from 10 cells ( n = 20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann–Whitney test ( P < 0.05). Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA‐BspF, separated into saponin‐, Triton X‐100–, and SDS‐soluble fractions and probed for HA‐BspF, Hsp27 (cytosol), Calnexin (membranes), and Lamin A/C (nucleus). Schematic depicting key host proteins that control transport pathways associated with the TGN‐RE‐plasma membrane compartment. Protein colors depict their compartmentalized functions. Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either GFP‐Rab6a, GFP‐Rab8a, or Arf6‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Quantification of localization of GFP‐Rab6a, GFP‐Rab8a, and Arf6‐GFP on mCherry‐BspF‐labeled tubules in transfected HeLa cells. Data are means ± SD from n = 3 independent experiments, in which at least 300 individual cells per experiment were analyzed. Source data are available online for this figure.

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Transfection, Expressing, Fluorescence, Staining, Software, MANN-WHITNEY, Western Blot, Labeling

    A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Transfection, Incubation, Binding Assay, Staining, Marker

    A Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry‐BspF (grayscale panels), incubated on ice with AlexaFluor™488‐Cholera Toxin subunit B (CTxB; green) and shifted to 37°C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). B Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry‐BspF, or HA‐BspF over a 30‐min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a two‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, and Rab6a/a′ depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA‐mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a), or Rab6a/a′ (siRab6a/a′) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with siNT control cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). E Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock‐infected or infected with wild‐type (2308), Δ bspF , complemented ∆ bspF (Δ bspF::bspF ), ∆ bspB or complemented ∆ bspB (Δ bspB::bspB ) bacteria for 24 h, incubated for 30 min with AlexaFluor™488‐CTxB on ice for binding followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with mock‐infected cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). F rBCV biogenesis in BMMs treated with either non‐targeting siNT, siRab6a/a′, or siRab8a siRNAs and infected with wild‐type (2308) bacteria. Data are means ± SD of n = 3 independent experiments, in which 100 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) compared with control (2308). G–I Brucella replication in BMMs treated with non‐targeting siRNAs (siNT), or siRNAs against Rab6a/a′ (siRab6a/a′) (G), Rab8a (siRab8a) (H), or Arf6 (siArf6) (I) and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: A Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry‐BspF (grayscale panels), incubated on ice with AlexaFluor™488‐Cholera Toxin subunit B (CTxB; green) and shifted to 37°C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). B Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry‐BspF, or HA‐BspF over a 30‐min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a two‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, and Rab6a/a′ depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA‐mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a), or Rab6a/a′ (siRab6a/a′) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with siNT control cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). E Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock‐infected or infected with wild‐type (2308), Δ bspF , complemented ∆ bspF (Δ bspF::bspF ), ∆ bspB or complemented ∆ bspB (Δ bspB::bspB ) bacteria for 24 h, incubated for 30 min with AlexaFluor™488‐CTxB on ice for binding followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared with mock‐infected cells as determined by a one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). F rBCV biogenesis in BMMs treated with either non‐targeting siNT, siRab6a/a′, or siRab8a siRNAs and infected with wild‐type (2308) bacteria. Data are means ± SD of n = 3 independent experiments, in which 100 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) compared with control (2308). G–I Brucella replication in BMMs treated with non‐targeting siRNAs (siNT), or siRNAs against Rab6a/a′ (siRab6a/a′) (G), Rab8a (siRab8a) (H), or Arf6 (siArf6) (I) and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Fluorescence, Transfection, Incubation, Staining, Marker, Western Blot, Binding Assay, Infection

    Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Transfection, Western Blot, Immunoprecipitation, Magnetic Beads, SDS Page, Activity Assay, Infection

    Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Fluorescence, Transfection, Labeling

    A Representative confocal micrographs of BMMs infected with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF::bspF ) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6‐positive vesicles and rBCVs. B Recruitment of Stx6‐positive vesicles to rBCVs (expressed as percentage of Stx6‐positive BCVs) in BMMs infected for 24 h with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF :: bspF ) bacteria. Data are means ± SD from n = 3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared with 2308‐infected BMMs as determined by one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, Rab6a/a′, and Stx6 depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D–F Recruitment of Stx6‐positive vesicles to rBCVs in BMMs treated with non‐targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E), or Rab6a/a′ (siRab6a/a′) (F) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions; ns, not significant. G Quantification of CTxB retrograde transport in BMMs following siRNA‐mediated depletion of Stx6 (siStx6) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference ( P = 0.0114, unpaired Student’s t ‐test) compared with the siNT control. H Brucella replication in BMMs treated with either non‐targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. I Model of BspF remodeling of TGN‐RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE‐TGN transport, which alters TGN‐derived vesicular traffic and redirects Stx6‐positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet: A Representative confocal micrographs of BMMs infected with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF::bspF ) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6‐positive vesicles and rBCVs. B Recruitment of Stx6‐positive vesicles to rBCVs (expressed as percentage of Stx6‐positive BCVs) in BMMs infected for 24 h with either wild‐type (2308), ∆ bspF , or complemented ∆ bspF (∆ bspF :: bspF ) bacteria. Data are means ± SD from n = 3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared with 2308‐infected BMMs as determined by one‐way ANOVA with Tukey’s multiple comparisons test ( P < 0.05). C Representative Western blot analysis of Arf6, Rab8a, Rab6a/a′, and Stx6 depletions in BMMs following siRNA‐mediated knockdowns, compared with non‐targeting siRNA (siNT) treatments. β‐actin was used as loading control. D–F Recruitment of Stx6‐positive vesicles to rBCVs in BMMs treated with non‐targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E), or Rab6a/a′ (siRab6a/a′) (F) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions; ns, not significant. G Quantification of CTxB retrograde transport in BMMs following siRNA‐mediated depletion of Stx6 (siStx6) after AlexaFluor™488‐CTxB binding on ice followed by 30‐min incubation at 37°C. Data are means ± SD from n = 3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference ( P = 0.0114, unpaired Student’s t ‐test) compared with the siNT control. H Brucella replication in BMMs treated with either non‐targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, one‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. I Model of BspF remodeling of TGN‐RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE‐TGN transport, which alters TGN‐derived vesicular traffic and redirects Stx6‐positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth. Source data are available online for this figure.

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Infection, Marker, Western Blot, Binding Assay, Incubation, Inhibition, Derivative Assay

    Journal: The EMBO Journal

    Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

    doi: 10.15252/embj.2021107664

    Figure Lengend Snippet:

    Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

    Techniques: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

    Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of Arf6 activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.

    Journal: eNeuro

    Article Title: ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus

    doi: 10.1523/ENEURO.0111-20.2020

    Figure Lengend Snippet: Generation of CentA1 KO mutant mice. A , Schematic drawing of the targeting strategy used to generate CentA1 global KO mouse lines. Exon 3 of Centa1 gene was replaced by sequences for LacZ, followed by a translational STOP, and a Neo selection cassette, flanked by frt sites. Expression and translation of this modified Centa1 locus resulted in a fusion protein of the beginning of CentA1 and LacZ, while functional CentA1 protein is lacking. B , NeuN immunohistochemistry of coronal sections from six- to eight-month-old CentA1 KO and NTG littermate mice show normal brain morphology in CentA1 KO mice. C , Immunoblots show the complete lack of CentA1 protein in the hippocampus from CentA1 KO mice. Bottom, Anti-β-Actin antibody shows that a similar amount of protein samples were loaded between genotypes. NTG: n = 5 mice; CentA1 KO: n = 3 mice. D , Immunoblots show that in the hippocampus of CentA1 KO mice, the level of another brain enriched Centaurin (Centγ3) does not undergo significant compensatory upregulation. The numbers under blots show hippocampal Centγ3 level normalized to β-Actin. NTG: n = 4 mice; CentA1 KO: n = 4 mice. Two-tailed t test, p = 0.29. E , Immunoblots show the level of Arf6 activation in the hippocampi of six-month-old CentA1 KO and NTG littermate mice, evaluated by active Arf6 pull-down assay. Bottom, Anti-Arf6 antibody shows a similar amount of total Arf6 protein in the hippocampal lysates between genotypes. Numbers under blots represent the level of active (GTP bound) Arf6 normalized to total Arf6 protein in the hippocampal tissue. NTG: n = 2 mice; CentA1 KO: n = 3 mice.

    Article Snippet: Membranes were blocked with 5% nonfat milk (Great Value) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies diluted in 5% BSA in TBS-T. We used the following commercially available antibodies: goat anti-Centaurin-ɑ1 (Abcam; 1:500), rabbit anti-Centaurin γ3 antibody (Santa Cruz Biotechnology; 1:500), mouse anti-β-Actin (Sigma, 1:1000); rabbit anti-Arf6 antibody (Cell Signaling Technologies; 1:1000), and mouse anti-Ras antibody (Thermo Fisher Scientific; 1:1000).

    Techniques: Mutagenesis, Selection, Expressing, Modification, Functional Assay, Immunohistochemistry, Western Blot, Two Tailed Test, Activation Assay, Pull Down Assay

    Endothelial cell Arf6 deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: Endothelial cell Arf6 deletion increases survival rates and reduces disease severity due to MDR A. baumannii pneumonia. (A) Survival of Arf6f/+ mice (n = 22) or endothelial cell ARF6 null mice (Arf6f/−;Tie2-cre) (n = 17) with A. baumannii pneumonia. (B) Lung bacterial burden (n = 12/arm); (C) lung permeability; (D) histopathological examination of lungs stained with H&E and harvested from Arf6f/+ or endothelial cell ARF6 null mice 4 days postinfection with A. baumannii via inhalation. Lungs from uninfected Arf6f/+ mice are included as a control. Black and white arrows in panel D denote tissue edema and hemorrhage in Arf6f/+ mice, respectively. Bars are 20 μM. Data shown in panels B and C are presented as the medians ± interquartile ranges.

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Permeability, Staining

    Pharmacologic inhibition of ARF6 increases survival rates and reduces disease severity due to MDR GNB infection. ARF6 inhibitors (A6-5093, NAV-2729, and A6-4424) increase survival (n = 20 mice for all groups, except those for A6-5093 [10 mice] and colistin [19 mice]) (A) and reduce lung bacterial burden (B) of immunosuppressed mice (200 mg/kg i.p. cyclophosphamide and 250 mg/kg subcutaneous cortisone acetate given on days −2 and +3 relative to infection) with MDR A. baumannii pneumonia. Lungs were harvested on day +4 relative to infection. *, P < 0.05 versus vehicle-treated mice; **, P < 0.02 versus all others in panel A; #, P < 0.05 versus vehicle-treated mice; ***, P < 0.03 versus all others in panel B. (C and D) ARF6 inhibitors increase survival rate of immunosuppressed mice infected with MDR CPKP (n = 10/group) (C) or MDR P. aeruginosa (n = 20/group) (D). Log rank test for all analyses of survival and Wilcoxon rank sum test for lung bacterial burden.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: Pharmacologic inhibition of ARF6 increases survival rates and reduces disease severity due to MDR GNB infection. ARF6 inhibitors (A6-5093, NAV-2729, and A6-4424) increase survival (n = 20 mice for all groups, except those for A6-5093 [10 mice] and colistin [19 mice]) (A) and reduce lung bacterial burden (B) of immunosuppressed mice (200 mg/kg i.p. cyclophosphamide and 250 mg/kg subcutaneous cortisone acetate given on days −2 and +3 relative to infection) with MDR A. baumannii pneumonia. Lungs were harvested on day +4 relative to infection. *, P < 0.05 versus vehicle-treated mice; **, P < 0.02 versus all others in panel A; #, P < 0.05 versus vehicle-treated mice; ***, P < 0.03 versus all others in panel B. (C and D) ARF6 inhibitors increase survival rate of immunosuppressed mice infected with MDR CPKP (n = 10/group) (C) or MDR P. aeruginosa (n = 20/group) (D). Log rank test for all analyses of survival and Wilcoxon rank sum test for lung bacterial burden.

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Inhibition, Infection

    ARF6 inhibition reduces organ permeability and disease severity without affecting the inflammatory response to A. baumannii pneumonia. (A) ARF6 inhibitors (NAV-2729 and A6-5093) decreased permeability in the lungs and kidneys of A. baumannii HUMC1-infected mice. ‡, P < 0.05 versus other treatments. (B) NAV-2729 (30 mg/kg) reduced severity of A. baumannii infection, as shown by histopathological examination of lungs with H&E stain and spleen with Gram stain. An asterisk denotes necrobiosis in spleen harvested from vehicle-treated mice and, to a lesser extent, colistin-treated mice, but not from mice treated with NAV-2729. Bars represent 100 μM. (C) IL-6 levels in lungs and plasma of A. baumannii HUMC1-infected mice and treated with vehicle control, NAV-2729, or colistin. §, P < 0.02 versus uninfected mice. Organs of neutropenic mice were harvested on day +3 (A) and day +4 (B and C) relative to infection. Data in panels A and C are presented as the medians ± interquartile ranges.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: ARF6 inhibition reduces organ permeability and disease severity without affecting the inflammatory response to A. baumannii pneumonia. (A) ARF6 inhibitors (NAV-2729 and A6-5093) decreased permeability in the lungs and kidneys of A. baumannii HUMC1-infected mice. ‡, P < 0.05 versus other treatments. (B) NAV-2729 (30 mg/kg) reduced severity of A. baumannii infection, as shown by histopathological examination of lungs with H&E stain and spleen with Gram stain. An asterisk denotes necrobiosis in spleen harvested from vehicle-treated mice and, to a lesser extent, colistin-treated mice, but not from mice treated with NAV-2729. Bars represent 100 μM. (C) IL-6 levels in lungs and plasma of A. baumannii HUMC1-infected mice and treated with vehicle control, NAV-2729, or colistin. §, P < 0.02 versus uninfected mice. Organs of neutropenic mice were harvested on day +3 (A) and day +4 (B and C) relative to infection. Data in panels A and C are presented as the medians ± interquartile ranges.

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Inhibition, Permeability, Infection, Staining

    A. baumannii-mediated HUVEC permeability is induced by LPS-TLR4 signaling through ARF6. (A) A. baumannii HUMC1 (virulent MDR) cells and supernatants (sup) induce HUVEC permeability to a level similar to that of E. coli LPS. Removing LPS by polymyxin B blocks this induction. ATCC 17978 (avirulent, drug-sensitive) cells and supernatants do not induce permeability. *, P < 0.005 versus HUMC1, HUMC1 supernatant, or E. coli LPS. (B) A permeability assay was conducted with HUMC1 or its supernatant in the presence of 50 μg/ml anti-TLR4 or isotype-matched control antibodies (Ab). *, P < 0.001 versus isotype-matched antibody. (C) ARF6-GTP pulldown assays show that A. baumannii HUMC1 (AB) induces ARF6 activation and NAV-2729 blocks it. Quantification of the ARF6-GTP/total ARF6 ratio by densitometer (3 independent experiments) shows A. baumannii induces endothelial cell ARF6 activation 2-fold, while NAV-2729 inhibits this activation. *, P < 0.05 versus all other comparators. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) A HUVEC permeability assay was conducted with HUMC1 in the presence of 50 μM ARF6 inhibitor (NAV-2729 or A6-4424). *, P < 0.0015 versus HUMC1 cells.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: A. baumannii-mediated HUVEC permeability is induced by LPS-TLR4 signaling through ARF6. (A) A. baumannii HUMC1 (virulent MDR) cells and supernatants (sup) induce HUVEC permeability to a level similar to that of E. coli LPS. Removing LPS by polymyxin B blocks this induction. ATCC 17978 (avirulent, drug-sensitive) cells and supernatants do not induce permeability. *, P < 0.005 versus HUMC1, HUMC1 supernatant, or E. coli LPS. (B) A permeability assay was conducted with HUMC1 or its supernatant in the presence of 50 μg/ml anti-TLR4 or isotype-matched control antibodies (Ab). *, P < 0.001 versus isotype-matched antibody. (C) ARF6-GTP pulldown assays show that A. baumannii HUMC1 (AB) induces ARF6 activation and NAV-2729 blocks it. Quantification of the ARF6-GTP/total ARF6 ratio by densitometer (3 independent experiments) shows A. baumannii induces endothelial cell ARF6 activation 2-fold, while NAV-2729 inhibits this activation. *, P < 0.05 versus all other comparators. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) A HUVEC permeability assay was conducted with HUMC1 in the presence of 50 μM ARF6 inhibitor (NAV-2729 or A6-4424). *, P < 0.0015 versus HUMC1 cells.

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Permeability, Activation Assay

    A. baumannii-mediated HUVEC permeability is induced through the MyD88/ARNO/ARF6 pathway. (A) Successful downregulation of gene expression (quantitative reverse transcription-PCR [qRT-PCR]) using siRNA constructs targeting ARF6, ARNO, or MyD88 in HUVECs. *, P < 0.001 versus untransfected HUVECs (2 experiments). (B and C) ARF6 expression as determined by qRT-PCR and Western blotting (B) and A. baumannii-mediated permeability using FITC-dextran (C) of HUVECs transfected with siRNA constructs targeting MyD88, ARNO, ARF6, or scrambled sequence (control). *, P < 0.008 versus control siRNA plus A. baumannii (B) and P < 0.002 versus control siRNA plus A. baumannii (C).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: A. baumannii-mediated HUVEC permeability is induced through the MyD88/ARNO/ARF6 pathway. (A) Successful downregulation of gene expression (quantitative reverse transcription-PCR [qRT-PCR]) using siRNA constructs targeting ARF6, ARNO, or MyD88 in HUVECs. *, P < 0.001 versus untransfected HUVECs (2 experiments). (B and C) ARF6 expression as determined by qRT-PCR and Western blotting (B) and A. baumannii-mediated permeability using FITC-dextran (C) of HUVECs transfected with siRNA constructs targeting MyD88, ARNO, ARF6, or scrambled sequence (control). *, P < 0.008 versus control siRNA plus A. baumannii (B) and P < 0.002 versus control siRNA plus A. baumannii (C).

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Permeability, Expressing, Quantitative RT-PCR, Construct, Western Blot, Transfection, Sequencing

    Model of GNB-induced vascular leak and the role of ARF6 inhibitor in preserving vascular integrity and increasing survival in bacterial infections. (A) In a normal host, the vasculature is held intact by VE-cadherin localized to the cell-cell junctions. (B) In bacterial sepsis (e.g., A. baumannii [AB]), LPS-induced endotoxemia triggers a robust host inflammatory response by TLR4-mediated activation of MyD88/NF-κB required for clearing the infection. (C) Bacterial LPS also triggers ARF6 activation via MyD88/ARNO, which leads to intercellular recruitment of VE-cadherin, resulting in increased vascular leak, tissue edema, organ failure, and, ultimately, death. (D) ARF6 inhibitors prevent ARF6 activation, which reduces VE-cadherin internalization, resulting in the preservation of vascular integrity without affecting the inflammatory immune response.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection

    doi: 10.1128/AAC.00303-20

    Figure Lengend Snippet: Model of GNB-induced vascular leak and the role of ARF6 inhibitor in preserving vascular integrity and increasing survival in bacterial infections. (A) In a normal host, the vasculature is held intact by VE-cadherin localized to the cell-cell junctions. (B) In bacterial sepsis (e.g., A. baumannii [AB]), LPS-induced endotoxemia triggers a robust host inflammatory response by TLR4-mediated activation of MyD88/NF-κB required for clearing the infection. (C) Bacterial LPS also triggers ARF6 activation via MyD88/ARNO, which leads to intercellular recruitment of VE-cadherin, resulting in increased vascular leak, tissue edema, organ failure, and, ultimately, death. (D) ARF6 inhibitors prevent ARF6 activation, which reduces VE-cadherin internalization, resulting in the preservation of vascular integrity without affecting the inflammatory immune response.

    Article Snippet: The excision of the Arf6 f allele was verified by isolating lung endothelial cells, as previously described ( 31 ), and performing Western blotting to assess ARF6 levels using anti-ARF6 antibody from Cell Signaling Technology (number 3546) (Fig. S4B). (ii) Infection with GNB.

    Techniques: Preserving, Activation Assay, Infection