cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cortactin
    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and <t>cortactin</t> (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.
    Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients"

    Article Title: High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-12-17

    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.
    Figure Legend Snippet: Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.

    Techniques Used: Immunohistochemical staining, Staining

    cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cortactin
    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and <t>cortactin</t> (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.
    Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients"

    Article Title: High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-12-17

    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.
    Figure Legend Snippet: Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.

    Techniques Used: Immunohistochemical staining, Staining

    cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cortactin
    (A) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +16∶0 fatty acid for 24 hours. (B) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +18∶1 fatty acid for 24 hours. (C) Invadopodia incidence in 3T3-Src cells transfected with 100 nM scrambled (Sc) siRNA or siRNA #1 against ACC1 for 48 hours and then supplemented with 18∶1 fatty acid for 24 hours. (D) ACC1 protein expression in 3T3-Src cells transfected with 100 nM scrambled or siRNA #1 against ACC1 and supplemented with 18∶1 fatty acid. (E) <t>Cortactin</t> localization to invadopodia in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA supplemented with 18∶1 fatty acid for 24 hours. Cortactin co-localization with F-actin was determined by immunofluorescence. Scale bar,1 µm. (F) 3T3-Src cells were treated with BSA or 18∶1 fatty acid for 24 hours and invadopodia incidence was determined. Scale bar, 25 µm *p≤0.05; **p≤0.01.
    Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis"

    Article Title: Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029761

    (A) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +16∶0 fatty acid for 24 hours. (B) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +18∶1 fatty acid for 24 hours. (C) Invadopodia incidence in 3T3-Src cells transfected with 100 nM scrambled (Sc) siRNA or siRNA #1 against ACC1 for 48 hours and then supplemented with 18∶1 fatty acid for 24 hours. (D) ACC1 protein expression in 3T3-Src cells transfected with 100 nM scrambled or siRNA #1 against ACC1 and supplemented with 18∶1 fatty acid. (E) Cortactin localization to invadopodia in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA supplemented with 18∶1 fatty acid for 24 hours. Cortactin co-localization with F-actin was determined by immunofluorescence. Scale bar,1 µm. (F) 3T3-Src cells were treated with BSA or 18∶1 fatty acid for 24 hours and invadopodia incidence was determined. Scale bar, 25 µm *p≤0.05; **p≤0.01.
    Figure Legend Snippet: (A) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +16∶0 fatty acid for 24 hours. (B) Invadopodia incidence in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA +18∶1 fatty acid for 24 hours. (C) Invadopodia incidence in 3T3-Src cells transfected with 100 nM scrambled (Sc) siRNA or siRNA #1 against ACC1 for 48 hours and then supplemented with 18∶1 fatty acid for 24 hours. (D) ACC1 protein expression in 3T3-Src cells transfected with 100 nM scrambled or siRNA #1 against ACC1 and supplemented with 18∶1 fatty acid. (E) Cortactin localization to invadopodia in 3T3-Src cells treated with vehicle, TOFA (30 µM), or TOFA supplemented with 18∶1 fatty acid for 24 hours. Cortactin co-localization with F-actin was determined by immunofluorescence. Scale bar,1 µm. (F) 3T3-Src cells were treated with BSA or 18∶1 fatty acid for 24 hours and invadopodia incidence was determined. Scale bar, 25 µm *p≤0.05; **p≤0.01.

    Techniques Used: Transfection, Expressing, Immunofluorescence

    cttn antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cttn antibody
    CSC inhibits both Cortactin and Survivin RNA expression levels parallel <t>to</t> <t>CD44</t> expression. (A) CSC-reduced CD44 RNA levels paralleled <t>CTTN</t> RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.
    Cttn Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CD44 mediates stem cell mobilization to damaged lung via its novel transcriptional targets, Cortactin and Survivin"

    Article Title: CD44 mediates stem cell mobilization to damaged lung via its novel transcriptional targets, Cortactin and Survivin

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.33125

    CSC inhibits both Cortactin and Survivin RNA expression levels parallel to CD44 expression. (A) CSC-reduced CD44 RNA levels paralleled CTTN RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.
    Figure Legend Snippet: CSC inhibits both Cortactin and Survivin RNA expression levels parallel to CD44 expression. (A) CSC-reduced CD44 RNA levels paralleled CTTN RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Techniques Used: RNA Expression, Expressing

    CSC inhibits both Cortactin and Survivin protein expression levels parallel to CD44 protein expression. (A) CSC-reduced CD44 protein expression levels paralleled CTTN protein expression levels. (B) CSC-reduced CD44 protein expression levels paralleled BIRC5 protein expression levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.
    Figure Legend Snippet: CSC inhibits both Cortactin and Survivin protein expression levels parallel to CD44 protein expression. (A) CSC-reduced CD44 protein expression levels paralleled CTTN protein expression levels. (B) CSC-reduced CD44 protein expression levels paralleled BIRC5 protein expression levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Techniques Used: Expressing

    α cortactin antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc α cortactin antibody
    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of <t>cortactin.</t> INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an <t>α-cortactin</t> antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    α Cortactin Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni"

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-82

    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Infection, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Transfection, Construct

    Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.
    Figure Legend Snippet: Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.

    Techniques Used: Construct, Transfection, Confocal Microscopy, Infection, Staining

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .
    Figure Legend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .

    Techniques Used: Transfection, Construct, Electron Microscopy, Infection

    CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Infection, Mutagenesis, Immunoprecipitation, SDS Page

    Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.
    Figure Legend Snippet: Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.

    Techniques Used: Binding Assay, Activation Assay

    cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cortactin
    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of <t>cortactin.</t> INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
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    1) Product Images from "Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni"

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-82

    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Infection, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Transfection, Construct

    Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.
    Figure Legend Snippet: Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.

    Techniques Used: Construct, Transfection, Confocal Microscopy, Infection, Staining

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .
    Figure Legend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .

    Techniques Used: Transfection, Construct, Electron Microscopy, Infection

    CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
    Figure Legend Snippet: CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Techniques Used: Infection, Mutagenesis, Immunoprecipitation, SDS Page

    Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.
    Figure Legend Snippet: Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.

    Techniques Used: Binding Assay, Activation Assay

    anti cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cortactin
    THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and <t>cortactin</t> in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.
    Anti Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The PSMD14 inhibitor Thiolutin as a novel therapeutic approach for esophageal squamous cell carcinoma through facilitating SNAIL degradation"

    Article Title: The PSMD14 inhibitor Thiolutin as a novel therapeutic approach for esophageal squamous cell carcinoma through facilitating SNAIL degradation

    Journal: Theranostics

    doi: 10.7150/thno.46109

    THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and cortactin in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.
    Figure Legend Snippet: THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and cortactin in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.

    Techniques Used: Transwell Assay, Migration, Western Blot, Immunofluorescence, Fluorescence

    rabbit polyclonal antibodies against cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibodies against cortactin
    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified <t>polyclonal</t> antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and <t>anti-cortactin</t> monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
    Rabbit Polyclonal Antibodies Against Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction"

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008478

    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
    Figure Legend Snippet: Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Techniques Used: Cell Culture, Labeling, Binding Assay, Affinity Purification

    Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see ), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).
    Figure Legend Snippet: Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see ), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).

    Techniques Used: Labeling, Cell Culture, Staining

    R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).
    Figure Legend Snippet: R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).

    Techniques Used: Labeling, Generated, Antibody Labeling

    Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see ), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.
    Figure Legend Snippet: Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see ), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.

    Techniques Used: Cell Culture, Immunoprecipitation, Staining

    To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.
    Figure Legend Snippet: To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.

    Techniques Used: Transfection, Mutagenesis, Expressing, Fluorescence, Labeling, Marker

    C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.
    Figure Legend Snippet: C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.

    Techniques Used: Generated, Transfection, Incubation, Labeling, Fluorescence, Expressing

    Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see ) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.
    Figure Legend Snippet: Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see ) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Labeling

    Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.
    Figure Legend Snippet: Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Techniques Used: Activation Assay

    antibodies against cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against cortactin
    Effects of HDAC6 on LPS-induced <t>cortactin</t> localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.
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    Images

    1) Product Images from "Histone deacetylase 6 modulates macrophage infiltration during inflammation"

    Article Title: Histone deacetylase 6 modulates macrophage infiltration during inflammation

    Journal: Theranostics

    doi: 10.7150/thno.25317

    Effects of HDAC6 on LPS-induced cortactin localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.
    Figure Legend Snippet: Effects of HDAC6 on LPS-induced cortactin localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.

    Techniques Used: Knock-Out

    Proposed model for HDAC6-mediated macrophage infiltration and phagocytosis. In wild-type unstimulated macrophages, HDAC6 localizes to the cytosol. In response to inflammatory cues, HDAC6 partially translocates to the cell periphery, where it deacetylates α-tubulin and cortactin, thereby enhancing dynamics of microtubule tips and promoting actin assembly. Changes in cytoskeletal dynamics promote macrophage infiltration and phagocytosis. In the absence of HDAC6, cytoskeletal dynamics are not affected, rendering macrophages insensitive to inflammatory cues.
    Figure Legend Snippet: Proposed model for HDAC6-mediated macrophage infiltration and phagocytosis. In wild-type unstimulated macrophages, HDAC6 localizes to the cytosol. In response to inflammatory cues, HDAC6 partially translocates to the cell periphery, where it deacetylates α-tubulin and cortactin, thereby enhancing dynamics of microtubule tips and promoting actin assembly. Changes in cytoskeletal dynamics promote macrophage infiltration and phagocytosis. In the absence of HDAC6, cytoskeletal dynamics are not affected, rendering macrophages insensitive to inflammatory cues.

    Techniques Used:

    anti cortactin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cortactin
    SIRT1 deacetylates <t>Cortactin</t> in corneal epithelial cells. ( A ) Diagrammatic representation of the workflow used to analyze the interacting proteins with SIRT1 in HCECs using mass spectrometry analysis. ( B ) Immunofluorescence evaluation of SIRT1 ( green ) and endogenous Cortactin ( red ) expression in HCEC. The nucleus was stained with DAPI ( blue ). Representative green, red, blue, and merge images (×400) captured on a confocal microscope are shown. Scale bars : 50 µm. ( C ) Representative images of the immunoprecipitation bands. After immunoprecipitation of cell lysates with anti-Sirt1 antibody, the immunoprecipitants were immunoblotted with an anti-Cortactin antibody. Immunoprecipitants with IgG were used as controls. ( D ) After immunoprecipitation of cell lysates with anti-Cortactin antibody, the immunoprecipitants were immunoblotted with an anti-SIRT1 antibody. Immunoprecipitants with IgG were used as controls. ( E ) Representative immunofluorescence images showed that the fluorescence intensity of Ac-cortactin in the siSIRT1 transfected HCECs was more potent compared with the control counterpart. Scale bars : 50 µm. ( F ) Western blotting analyzed the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected HCECs. ( G ) Quantification of densitometry of the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected cells (n = 3/group). ( H ) Representative immunofluorescence images of mouse corneal cryosections and statistical analysis showed the fluorescence intensity of Ac-cortactin in the Sirt1 cKO was stronger compared with the control counterparts ( Krt12-cre + and Sirt1 flox/flox mice), while there is no alteration of the intensity of Cortactin (n = 6/group). ( I ) Western blotting analyzed protein levels of Ac-cortactin and the total cortactin in the cKO corneal epithelia and the control corneas, and the densitometry of the protein levels of Ac-Cortactin and the total Cortactin was quantified (n = 3/group).
    Anti Cortactin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Sirt1 Regulates Corneal Epithelial Migration by Deacetylating Cortactin"

    Article Title: Sirt1 Regulates Corneal Epithelial Migration by Deacetylating Cortactin

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.63.12.14

    SIRT1 deacetylates Cortactin in corneal epithelial cells. ( A ) Diagrammatic representation of the workflow used to analyze the interacting proteins with SIRT1 in HCECs using mass spectrometry analysis. ( B ) Immunofluorescence evaluation of SIRT1 ( green ) and endogenous Cortactin ( red ) expression in HCEC. The nucleus was stained with DAPI ( blue ). Representative green, red, blue, and merge images (×400) captured on a confocal microscope are shown. Scale bars : 50 µm. ( C ) Representative images of the immunoprecipitation bands. After immunoprecipitation of cell lysates with anti-Sirt1 antibody, the immunoprecipitants were immunoblotted with an anti-Cortactin antibody. Immunoprecipitants with IgG were used as controls. ( D ) After immunoprecipitation of cell lysates with anti-Cortactin antibody, the immunoprecipitants were immunoblotted with an anti-SIRT1 antibody. Immunoprecipitants with IgG were used as controls. ( E ) Representative immunofluorescence images showed that the fluorescence intensity of Ac-cortactin in the siSIRT1 transfected HCECs was more potent compared with the control counterpart. Scale bars : 50 µm. ( F ) Western blotting analyzed the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected HCECs. ( G ) Quantification of densitometry of the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected cells (n = 3/group). ( H ) Representative immunofluorescence images of mouse corneal cryosections and statistical analysis showed the fluorescence intensity of Ac-cortactin in the Sirt1 cKO was stronger compared with the control counterparts ( Krt12-cre + and Sirt1 flox/flox mice), while there is no alteration of the intensity of Cortactin (n = 6/group). ( I ) Western blotting analyzed protein levels of Ac-cortactin and the total cortactin in the cKO corneal epithelia and the control corneas, and the densitometry of the protein levels of Ac-Cortactin and the total Cortactin was quantified (n = 3/group).
    Figure Legend Snippet: SIRT1 deacetylates Cortactin in corneal epithelial cells. ( A ) Diagrammatic representation of the workflow used to analyze the interacting proteins with SIRT1 in HCECs using mass spectrometry analysis. ( B ) Immunofluorescence evaluation of SIRT1 ( green ) and endogenous Cortactin ( red ) expression in HCEC. The nucleus was stained with DAPI ( blue ). Representative green, red, blue, and merge images (×400) captured on a confocal microscope are shown. Scale bars : 50 µm. ( C ) Representative images of the immunoprecipitation bands. After immunoprecipitation of cell lysates with anti-Sirt1 antibody, the immunoprecipitants were immunoblotted with an anti-Cortactin antibody. Immunoprecipitants with IgG were used as controls. ( D ) After immunoprecipitation of cell lysates with anti-Cortactin antibody, the immunoprecipitants were immunoblotted with an anti-SIRT1 antibody. Immunoprecipitants with IgG were used as controls. ( E ) Representative immunofluorescence images showed that the fluorescence intensity of Ac-cortactin in the siSIRT1 transfected HCECs was more potent compared with the control counterpart. Scale bars : 50 µm. ( F ) Western blotting analyzed the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected HCECs. ( G ) Quantification of densitometry of the protein levels of Ac-cortactin and the total cortactin in the siSIRT1 transfected cells (n = 3/group). ( H ) Representative immunofluorescence images of mouse corneal cryosections and statistical analysis showed the fluorescence intensity of Ac-cortactin in the Sirt1 cKO was stronger compared with the control counterparts ( Krt12-cre + and Sirt1 flox/flox mice), while there is no alteration of the intensity of Cortactin (n = 6/group). ( I ) Western blotting analyzed protein levels of Ac-cortactin and the total cortactin in the cKO corneal epithelia and the control corneas, and the densitometry of the protein levels of Ac-Cortactin and the total Cortactin was quantified (n = 3/group).

    Techniques Used: Mass Spectrometry, Immunofluorescence, Expressing, Staining, Microscopy, Immunoprecipitation, Fluorescence, Transfection, Western Blot

    Cortactin decline elicits the same phenotype as SIRT1 ablation in the corneal epithelia. (A) Representative phase-contrast images of the initial wound (0 h) and at 24 hours after scratch wound in the siCortactin transfected and the control HCECs. Scale bars : 200 µm. ( B ) Quantification of the percentage of the initial wound area at 24 h after a scratch wound (n = 3/group). ( C ) Representative photographs and ( D ) Quantification of corneal wound areas in the siCortactin injected corneas and the control corneas at zero hours and 24 hours after corneal epithelial abrading (n = 6/group). (E) FITC-phalloidin ( green ) staining of cells at the wound margin showed polymerization of F-actin and formation of lamellipodia ( arrows ) along the wound edge. Cells were stained with FITC-phalloidin (green) to detect actin filaments, with DAPI ( blue ) to detect nuclei. Scale bars : 50 µm.
    Figure Legend Snippet: Cortactin decline elicits the same phenotype as SIRT1 ablation in the corneal epithelia. (A) Representative phase-contrast images of the initial wound (0 h) and at 24 hours after scratch wound in the siCortactin transfected and the control HCECs. Scale bars : 200 µm. ( B ) Quantification of the percentage of the initial wound area at 24 h after a scratch wound (n = 3/group). ( C ) Representative photographs and ( D ) Quantification of corneal wound areas in the siCortactin injected corneas and the control corneas at zero hours and 24 hours after corneal epithelial abrading (n = 6/group). (E) FITC-phalloidin ( green ) staining of cells at the wound margin showed polymerization of F-actin and formation of lamellipodia ( arrows ) along the wound edge. Cells were stained with FITC-phalloidin (green) to detect actin filaments, with DAPI ( blue ) to detect nuclei. Scale bars : 50 µm.

    Techniques Used: Transfection, Injection, Staining

    Schematic representation of SIRT1 regulation through deacetylating Cortactin during corneal epithelial migration.
    Figure Legend Snippet: Schematic representation of SIRT1 regulation through deacetylating Cortactin during corneal epithelial migration.

    Techniques Used: Migration

    cortactin  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc cortactin
    A Representative images of MF and IQGAP1 or IQGAP2 distribution in the directional migration model or after CXCL12 treatment. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. B Representative images of MF and IQGAP1 or IQGAP2 distribution in the wound healing assay. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. C Western blotting analysis of <t>Cortactin,</t> IQGAP1, and IQGAP2 levels in the NC, IQGAP1 -siRNA, IQGAP 2-siRNA, and IQGAP2 and IQGAP2 rescue groups. D Western blotting analysis of cortactin levels in the WT, S311A, S311D, S-A, and S-D groups. E Western blotting analysis of cortactin levels in the control, NC, RSK- siRNA, and RSK inhibitor treatment groups. F Representative images of filopodia and lamellipodia structures in the Clip170 mutation, and NC, IQGAP1 and IQGAP2 -siRNA groups (scale bar, 10 μm, green, FITC-stained MFs; blue, nucleus; white arrows: filopodia and lamellipodia structures).
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    Images

    1) Product Images from "Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration"

    Article Title: Tension of plus-end tracking protein Clip170 confers directionality and aggressiveness during breast cancer migration

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05306-6

    A Representative images of MF and IQGAP1 or IQGAP2 distribution in the directional migration model or after CXCL12 treatment. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. B Representative images of MF and IQGAP1 or IQGAP2 distribution in the wound healing assay. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. C Western blotting analysis of Cortactin, IQGAP1, and IQGAP2 levels in the NC, IQGAP1 -siRNA, IQGAP 2-siRNA, and IQGAP2 and IQGAP2 rescue groups. D Western blotting analysis of cortactin levels in the WT, S311A, S311D, S-A, and S-D groups. E Western blotting analysis of cortactin levels in the control, NC, RSK- siRNA, and RSK inhibitor treatment groups. F Representative images of filopodia and lamellipodia structures in the Clip170 mutation, and NC, IQGAP1 and IQGAP2 -siRNA groups (scale bar, 10 μm, green, FITC-stained MFs; blue, nucleus; white arrows: filopodia and lamellipodia structures).
    Figure Legend Snippet: A Representative images of MF and IQGAP1 or IQGAP2 distribution in the directional migration model or after CXCL12 treatment. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. B Representative images of MF and IQGAP1 or IQGAP2 distribution in the wound healing assay. (green, FITC-stained MFs; red, TRITC-stained IQGAP1/IQGAP2), scale bar, 10 μm. C Western blotting analysis of Cortactin, IQGAP1, and IQGAP2 levels in the NC, IQGAP1 -siRNA, IQGAP 2-siRNA, and IQGAP2 and IQGAP2 rescue groups. D Western blotting analysis of cortactin levels in the WT, S311A, S311D, S-A, and S-D groups. E Western blotting analysis of cortactin levels in the control, NC, RSK- siRNA, and RSK inhibitor treatment groups. F Representative images of filopodia and lamellipodia structures in the Clip170 mutation, and NC, IQGAP1 and IQGAP2 -siRNA groups (scale bar, 10 μm, green, FITC-stained MFs; blue, nucleus; white arrows: filopodia and lamellipodia structures).

    Techniques Used: Migration, Staining, Wound Healing Assay, Western Blot, Mutagenesis

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    Cell Signaling Technology Inc cortactin
    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and <t>cortactin</t> (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.
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    Cell Signaling Technology Inc cttn antibody
    CSC inhibits both Cortactin and Survivin RNA expression levels parallel <t>to</t> <t>CD44</t> expression. (A) CSC-reduced CD44 RNA levels paralleled <t>CTTN</t> RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.
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    Cell Signaling Technology Inc α cortactin antibody
    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of <t>cortactin.</t> INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an <t>α-cortactin</t> antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.
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    Cell Signaling Technology Inc anti cortactin
    THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and <t>cortactin</t> in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.
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    Cell Signaling Technology Inc rabbit polyclonal antibodies against cortactin
    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified <t>polyclonal</t> antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and <t>anti-cortactin</t> monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
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    Cell Signaling Technology Inc antibodies against cortactin
    Effects of HDAC6 on LPS-induced <t>cortactin</t> localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.
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    Image Search Results


    Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.

    Journal: Cell Communication and Signaling : CCS

    Article Title: High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients

    doi: 10.1186/1478-811X-12-17

    Figure Lengend Snippet: Immunohistochemical staining of EGFR (A), GEP100 (B), AMAP1 (C), and cortactin (D) in primary HNSCCs. Tissue sections were immunostained with antibodies against each target protein, as indicated. Positive staining of the proteins is shown in a reddish-brown color. The staining intensity of each protein in tumor cells was graded on a scale of 0–2, as described under Materials and methods . A representative figure for each staining is shown. Bars, 100 μm.

    Article Snippet: Antibodies against the following proteins were purchased from commercial sources: cortactin (CST, Lake Placid, NY) and EGFR (TDL and 31G7 mAb, Nichirei, Tokyo).

    Techniques: Immunohistochemical staining, Staining

    CSC inhibits both Cortactin and Survivin RNA expression levels parallel to CD44 expression. (A) CSC-reduced CD44 RNA levels paralleled CTTN RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Journal: International Journal of Medical Sciences

    Article Title: CD44 mediates stem cell mobilization to damaged lung via its novel transcriptional targets, Cortactin and Survivin

    doi: 10.7150/ijms.33125

    Figure Lengend Snippet: CSC inhibits both Cortactin and Survivin RNA expression levels parallel to CD44 expression. (A) CSC-reduced CD44 RNA levels paralleled CTTN RNA expression levels. (B) CSC-reduced CD44 RNA levels paralleled BIRC5 RNA levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Article Snippet: Membranes were probed with mouse anti-CD44 (1:2000 dilution, R& D systems), mouse monoclonal anti CTTN antibody (1:2000 dilution, Upstate cell signaling solution), rabbit monoclonal anti BIRC5 antibody (1:2000 dilution, Santa Cruz Biotechnology)and goat anti-GAPDH antibody (1:2000 dilution, Santa Cruz, CA) and detected with a goat anti-mouse and donkey anti-goat IgG-HRP (1:5000 dilution, Santa Cruz, CA) using chemiluminescence (Supersignal West Femto, Pierce).

    Techniques: RNA Expression, Expressing

    CSC inhibits both Cortactin and Survivin protein expression levels parallel to CD44 protein expression. (A) CSC-reduced CD44 protein expression levels paralleled CTTN protein expression levels. (B) CSC-reduced CD44 protein expression levels paralleled BIRC5 protein expression levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Journal: International Journal of Medical Sciences

    Article Title: CD44 mediates stem cell mobilization to damaged lung via its novel transcriptional targets, Cortactin and Survivin

    doi: 10.7150/ijms.33125

    Figure Lengend Snippet: CSC inhibits both Cortactin and Survivin protein expression levels parallel to CD44 protein expression. (A) CSC-reduced CD44 protein expression levels paralleled CTTN protein expression levels. (B) CSC-reduced CD44 protein expression levels paralleled BIRC5 protein expression levels. Shown are the cropped blot images representing indicated proteins, and all the gels from three separate experiments have been run under the same experimental conditions.

    Article Snippet: Membranes were probed with mouse anti-CD44 (1:2000 dilution, R& D systems), mouse monoclonal anti CTTN antibody (1:2000 dilution, Upstate cell signaling solution), rabbit monoclonal anti BIRC5 antibody (1:2000 dilution, Santa Cruz Biotechnology)and goat anti-GAPDH antibody (1:2000 dilution, Santa Cruz, CA) and detected with a goat anti-mouse and donkey anti-goat IgG-HRP (1:5000 dilution, Santa Cruz, CA) using chemiluminescence (Supersignal West Femto, Pierce).

    Techniques: Expressing

    Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Infection, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni -invasion of INT 407 cells. A . Internalization of C. jejuni in INT 407 cells transfected with siRNA to cortactin, siRNA to N-WASP or a scrambled (Scram) siRNA. Results are shown as the mean number of internalized bacteria ± SEM. B . Whole cell lysates of untreated, siRNA to cortactin, siRNA to N-WASP, and scramble siRNA-transfected cells were probed with α-cortactin and α-N-WASP antibodies. The blot was re-probed with an α-tubulin antibody to confirm equal loading. C . Internalization of C. jejuni in INT 407 cells transfected with phosphorylation null constructs of cortactin. Bacterial invasion was assessed using the gentamicin protection assay. Results are displayed as mean number of internalized bacteria ± SEM. D . Whole cell lysates of untreated and cortactin phosphorylation null transfected cells were collected and probed with an α-EGFP antibody. The blot was re-probed with an α-tubulin antibody to determine loading. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Transfection, Construct

    Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: Phosphorylation null constructs of cortactin prevent C. jejuni induced membrane ruffling. A-T . C. jejuni induced membrane ruffling is impaired in INT 407 cells transfected with cortactin S405A, S418A or S405/418A phosphorylation null constructs. Representative confocal microscopy images of INT 407 cells uninfected (Panel A-D ) and cells infected with the C. jejuni wild-type strain with various treatment conditions. The panels represent: Wild-type cortactin-EGFP (Panel E-H ), cortactin S405A phosphorylation null construct (Panel I-L ), cortactin S418A phosphorylation null construct (Panel M-P ), and cortactin S405/418A phosphorylation null construct (Panel Q-T ). Images from left to right show, DAPI staining of cell nuclei (Panels A , E , I , M , and Q ), EGFP-cortactin (Panel B , F , J , N , and R ), C. jejuni staining with a polyclonal rabbit α- Campylobacter antibody and a secondary Texas-Red antibody (Panels C , G , K , O , and S ), and merge of all panels (Panels D , H , L , P , and T ). INT 407 cells that display extensive membrane ruffling (Panel H ), and cells that display no host cell membrane ruffling (Panels D , L , P , and T ). C. jejuni in contact with the host cell is shown in (Panels H-1 , L-1 , P-1 , and T-1 ). Images were obtained with a 63× objective and have a 10 μM scale bar (Panels A-T ). Arrows indicate C. jejuni interaction with host cells . The areas within the box highlights regions of membrane ruffling and the areas within the circles indicate regions of no membrane ruffling.

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Construct, Transfection, Confocal Microscopy, Infection, Staining

    Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: Knockdown of endogenous cortactin and N-WASP prevent C. jejuni induced membrane ruffling. A-P . C. jejuni induced membrane ruffling in INT 407 cells transfected with scrambled (Scram) siRNA, siRNA to N-WASP, siRNA to cortactin, and cortactin S405A, S418A and S405/418A phosphorylation null constructs. Representative scanning electron microscopy images of INT 407 cells uninfected (Panel A ) and cells infected with C. jejuni wild-type strain with various treatment conditions; No treatment (Panel B ), Scrambled siRNA control (Panel C ), siRNA to N-WASP (Panel D ), siRNA to cortactin (Panel E ), cortactin S405A (Panel F ), cortactin S418A (Panel G ), and cortactin S405/4118A (Panel H ). Arrows in the higher magnification images show C. jejuni in contact with host cells (Panels I-P ). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. INT 407 cells that display extensive membrane ruffling (Panel J and K ), and INT 407 cells that display no host cell membrane ruffling (Panels L-P ). Images are shown at a magnification of 7,000× with a 10 μM scale bar (Panels A-H ), and 50,000× with a 2 μM scale bar (Panels I-P ). Also indicated within each panel is the percent of host cell that display membrane ruffling .

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Transfection, Construct, Electron Microscopy, Infection

    CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P < 0.01) compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis. N.S. indicates no significant difference.

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Infection, Mutagenesis, Immunoprecipitation, SDS Page

    Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.

    Journal: Cell Communication and Signaling : CCS

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    doi: 10.1186/1478-811X-11-82

    Figure Lengend Snippet: Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ , ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ , ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ , ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ , , , ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ , , ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.

    Article Snippet: Immunoprecipitations were performed by incubating cell lysates with an α-cortactin antibody (Cell Signaling Technology, Inc., Danvers, MA) at 4°C overnight and then adding protein A/G beads at 4°C for 1 h with rotation.

    Techniques: Binding Assay, Activation Assay

    THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and cortactin in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: Theranostics

    Article Title: The PSMD14 inhibitor Thiolutin as a novel therapeutic approach for esophageal squamous cell carcinoma through facilitating SNAIL degradation

    doi: 10.7150/thno.46109

    Figure Lengend Snippet: THL undermines cell motility and reverses EMT process. (A) Transwell assay was performed to show that THL attenuated significantly migration and invasion abilities of KYSE 30 and KYSE 150 cells. Scale bars, 100 μm. (B) The proteins of E-Cadherin, N-Cadherin, Vimentin and SNAIL were determined by immunoblotting in indicated treated ESCC cells. (C) The level of E-Cadherin was assessed by immunofluorescence in KYSE 30 and KYSE 150 cells treated with THL or DMSO for 24 h. Scale bar, 20 μm. (D) Immunofluorescence of F-actin and cortactin in DMSO and THL-treated cells. Scale bar, 20 μm. (E) The Fluorescence intensities of F-actin and cortactin along with the yellow lines marked in (D) . Data in this figure, mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: The antibodies used in this study were as follows: anti-PSMD14 (12059-1-AP) and anti-SNAIL (13099-1-AP) were purchased from Proteintech (Rosemont, IL, USA); anti-PSMD14 (HPA002114) was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-PSMD14 (sc-100464) and anti-GAPDH (sc-365062) were obtained from Santa Cruz (Dallas, Texas, USA); anti-SNAIL (#3879), anti-E-Cadherin (#3195), anti-N-Cadherin (#13116), anti-Vimentin (#5741), anti-Cortactin (#3503), anti-PARP (#9542), anti-Caspase-3 (#9662), anti-Caspase-9 (#9502), anti-Ubiquitin (#3936) and Normal Rabbit IgG (#2729) were all from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Transwell Assay, Migration, Western Blot, Immunofluorescence, Fluorescence

    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Cell Culture, Labeling, Binding Assay, Affinity Purification

    Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see ), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see ), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Labeling, Cell Culture, Staining

    R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Labeling, Generated, Antibody Labeling

    Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see ), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see ), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Cell Culture, Immunoprecipitation, Staining

    To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Transfection, Mutagenesis, Expressing, Fluorescence, Labeling, Marker

    C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Generated, Transfection, Incubation, Labeling, Fluorescence, Expressing

    Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see ) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see ) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Expressing, Mutagenesis, Cell Culture, Labeling

    Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Activation Assay

    Effects of HDAC6 on LPS-induced cortactin localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.

    Journal: Theranostics

    Article Title: Histone deacetylase 6 modulates macrophage infiltration during inflammation

    doi: 10.7150/thno.25317

    Figure Lengend Snippet: Effects of HDAC6 on LPS-induced cortactin localization and filopodial protrusion formation. (A) Macrophages from wild-type mice were treated with PBS or LPS and immunostained with antibodies against HDAC6 and cortactin. Arrowheads indicate the cell membrane. Scale bar, 20 μm. (B) Macrophages from HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin and cortactin. Scale bar, 20 μm. (C) Macrophages from wild-type or HDAC6 knockout mice were treated with PBS or LPS and immunostained with antibodies against F-actin. Scale bar, 20 μm. (D) Experiments were performed as in (C), and the percentages of cells with filopodial protrusions were quantified. ***P < 0.001; ns, not significant.

    Article Snippet: Antibodies against cortactin (Cell Signaling Technology), α-tubulin (Abcam), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), and fluorescein- or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were obtained from the indicated sources.

    Techniques: Knock-Out

    Proposed model for HDAC6-mediated macrophage infiltration and phagocytosis. In wild-type unstimulated macrophages, HDAC6 localizes to the cytosol. In response to inflammatory cues, HDAC6 partially translocates to the cell periphery, where it deacetylates α-tubulin and cortactin, thereby enhancing dynamics of microtubule tips and promoting actin assembly. Changes in cytoskeletal dynamics promote macrophage infiltration and phagocytosis. In the absence of HDAC6, cytoskeletal dynamics are not affected, rendering macrophages insensitive to inflammatory cues.

    Journal: Theranostics

    Article Title: Histone deacetylase 6 modulates macrophage infiltration during inflammation

    doi: 10.7150/thno.25317

    Figure Lengend Snippet: Proposed model for HDAC6-mediated macrophage infiltration and phagocytosis. In wild-type unstimulated macrophages, HDAC6 localizes to the cytosol. In response to inflammatory cues, HDAC6 partially translocates to the cell periphery, where it deacetylates α-tubulin and cortactin, thereby enhancing dynamics of microtubule tips and promoting actin assembly. Changes in cytoskeletal dynamics promote macrophage infiltration and phagocytosis. In the absence of HDAC6, cytoskeletal dynamics are not affected, rendering macrophages insensitive to inflammatory cues.

    Article Snippet: Antibodies against cortactin (Cell Signaling Technology), α-tubulin (Abcam), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), and fluorescein- or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were obtained from the indicated sources.

    Techniques: