rabbit anti py118 paxillin  (Cell Signaling Technology Inc)


Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit anti py118 paxillin
    Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous <t>pY118-Paxillin</t> staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .
    Rabbit Anti Py118 Paxillin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti py118 paxillin/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti py118 paxillin - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Lack of Paxillin phosphorylation promotes single-cell migration in vivo"

    Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202206078

    Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .
    Figure Legend Snippet: Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Techniques Used: In Vivo, In Vitro, Cell Culture, Sequencing, Staining, Immunostaining, Transplantation Assay, Western Blot, Expressing, Migration

    Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .
    Figure Legend Snippet: Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .

    Techniques Used: In Vivo, In Vitro, Immunostaining, Cell Culture, Transplantation Assay, Western Blot, Staining, Expressing

    Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.
    Figure Legend Snippet: Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

    Techniques Used: Expressing, In Vivo, Immunostaining, Imaging, Migration, Cell Tracking Assay

    FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .
    Figure Legend Snippet: FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Techniques Used: In Vivo, In Vitro, Cell Culture, Activation Assay, Migration, Western Blot, Expressing, Immunoprecipitation

    rabbit anti paxillin py118  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit anti paxillin py118
    PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin <t>pY118</t> followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.
    Rabbit Anti Paxillin Py118, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti paxillin py118/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti paxillin py118 - by Bioz Stars, 2024-07
    96/100 stars

    Images

    1) Product Images from "Prosaposin down-modulation decreases metastatic prostate cancer cell adhesion, migration, and invasion"

    Article Title: Prosaposin down-modulation decreases metastatic prostate cancer cell adhesion, migration, and invasion

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-30

    PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin pY118 followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.
    Figure Legend Snippet: PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin pY118 followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.

    Techniques Used: Activity Assay, Incubation, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

    rabbit anti py118 paxillin  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc rabbit anti py118 paxillin
    (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Upper panel: <t>pY118-Paxillin</t> staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Lower panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 days post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin staining. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes and YUMM1.7 melanoma in vivo tumors. In vitro and in vivo bands are from the same blot – see entire blot in . GFP was used as the loading control and a control for number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from (C). Non-parametric unpaired t test, *p<0.05. n=3 individual experiments. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions (n=64 cells for WT, n=32 cells for Y118E and n=35 cells for Y118F) and in vivo (n=8 cells/3 fish for WT, n=12 cells/3 fish for Y118E and n=15 cells/3 fish for Y118F). Larval zebrafish are imaged at 1 day post-transplantation. Non-parametric one-way ANOVA, *p<0.05, ****p<0.0001. Error bars are mean ± SD.
    Rabbit Anti Py118 Paxillin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti py118 paxillin/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti py118 paxillin - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation"

    Article Title: Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

    Journal: bioRxiv

    doi: 10.1101/2022.03.02.482703

    (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Upper panel: pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Lower panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 days post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin staining. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes and YUMM1.7 melanoma in vivo tumors. In vitro and in vivo bands are from the same blot – see entire blot in . GFP was used as the loading control and a control for number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from (C). Non-parametric unpaired t test, *p<0.05. n=3 individual experiments. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions (n=64 cells for WT, n=32 cells for Y118E and n=35 cells for Y118F) and in vivo (n=8 cells/3 fish for WT, n=12 cells/3 fish for Y118E and n=15 cells/3 fish for Y118F). Larval zebrafish are imaged at 1 day post-transplantation. Non-parametric one-way ANOVA, *p<0.05, ****p<0.0001. Error bars are mean ± SD.
    Figure Legend Snippet: (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Upper panel: pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Lower panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 days post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin staining. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes and YUMM1.7 melanoma in vivo tumors. In vitro and in vivo bands are from the same blot – see entire blot in . GFP was used as the loading control and a control for number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from (C). Non-parametric unpaired t test, *p<0.05. n=3 individual experiments. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions (n=64 cells for WT, n=32 cells for Y118E and n=35 cells for Y118F) and in vivo (n=8 cells/3 fish for WT, n=12 cells/3 fish for Y118E and n=15 cells/3 fish for Y118F). Larval zebrafish are imaged at 1 day post-transplantation. Non-parametric one-way ANOVA, *p<0.05, ****p<0.0001. Error bars are mean ± SD.

    Techniques Used: Sequencing, Staining, Immunostaining, In Vitro, Cell Culture, Transplantation Assay, Western Blot, Expressing, In Vivo, Migration

    (A) Upper panel: pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 days post-transplantation). Lower panel: pY118-Paxillin staining (magenta) of zebrafish developing heart (5dpf). (B) pY118-Paxillin staining of ZMEL cells expressing wildtype Paxillin plated on collagen coated dishes with different stiffness: 0.5kPa (upper panel), 50kPa (middle panel), and glass bottom dishes (lower panel). Scale bar is 10 µm. (C) Entire western blot of panels shown in – YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors ( in vivo ) blotted with Paxillin and pY118-Paxillin antibodies. GFP was used as the loading control, and as a control for the number of YUMM1.7 cells in mouse tumors.
    Figure Legend Snippet: (A) Upper panel: pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 days post-transplantation). Lower panel: pY118-Paxillin staining (magenta) of zebrafish developing heart (5dpf). (B) pY118-Paxillin staining of ZMEL cells expressing wildtype Paxillin plated on collagen coated dishes with different stiffness: 0.5kPa (upper panel), 50kPa (middle panel), and glass bottom dishes (lower panel). Scale bar is 10 µm. (C) Entire western blot of panels shown in – YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors ( in vivo ) blotted with Paxillin and pY118-Paxillin antibodies. GFP was used as the loading control, and as a control for the number of YUMM1.7 cells in mouse tumors.

    Techniques Used: Staining, Immunostaining, In Vitro, Cell Culture, Transplantation Assay, Expressing, Western Blot, In Vivo

    (A) pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg(mpeg:Lifeact-GFP) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin staining of a non-macrophage cell. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg(mpeg:WT-Paxillin-EGFP) zj503 , Tg(mpeg: Y118E-Paxillin-EGFP) zj504 and Tg(mpeg:Y118F-Paxillin-EGFP) zj505 embryos at timepoint 0 and 10 mins. Dotted lines indicate wound sites and arrows show the direction of migration. See also supplemental video S7. Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo . Error bars are mean ± SD. Non-parametric one-way ANOVA, ****p<0.0001. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo , migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x-axis (n=38 cells/6 fish for WT, n=20 cells/6 fish for Y118E and n=24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.
    Figure Legend Snippet: (A) pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg(mpeg:Lifeact-GFP) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin staining of a non-macrophage cell. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg(mpeg:WT-Paxillin-EGFP) zj503 , Tg(mpeg: Y118E-Paxillin-EGFP) zj504 and Tg(mpeg:Y118F-Paxillin-EGFP) zj505 embryos at timepoint 0 and 10 mins. Dotted lines indicate wound sites and arrows show the direction of migration. See also supplemental video S7. Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo . Error bars are mean ± SD. Non-parametric one-way ANOVA, ****p<0.0001. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo , migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x-axis (n=38 cells/6 fish for WT, n=20 cells/6 fish for Y118E and n=24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

    Techniques Used: Immunostaining, Staining, Imaging, Migration, In Vivo, Cell Tracking Assay

    anti py118 paxillin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti py118 paxillin
    Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous <t>pY118-Paxillin</t> staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .
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    PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin <t>pY118</t> followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.
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    PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin <t>pY118</t> followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.
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    Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

    doi: 10.1083/jcb.202206078

    Figure Lengend Snippet: Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

    Techniques: In Vivo, In Vitro, Cell Culture, Sequencing, Staining, Immunostaining, Transplantation Assay, Western Blot, Expressing, Migration

    Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

    doi: 10.1083/jcb.202206078

    Figure Lengend Snippet: Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .

    Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

    Techniques: In Vivo, In Vitro, Immunostaining, Cell Culture, Transplantation Assay, Western Blot, Staining, Expressing

    Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

    Journal: The Journal of Cell Biology

    Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

    doi: 10.1083/jcb.202206078

    Figure Lengend Snippet: Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

    Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

    Techniques: Expressing, In Vivo, Immunostaining, Imaging, Migration, Cell Tracking Assay

    FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

    doi: 10.1083/jcb.202206078

    Figure Lengend Snippet: FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

    Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

    Techniques: In Vivo, In Vitro, Cell Culture, Activation Assay, Migration, Western Blot, Expressing, Immunoprecipitation

    PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin pY118 followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.

    Journal: Molecular Cancer

    Article Title: Prosaposin down-modulation decreases metastatic prostate cancer cell adhesion, migration, and invasion

    doi: 10.1186/1476-4598-9-30

    Figure Lengend Snippet: PSAP down-modulation decreased FAK activity and prevented β 1A -integrin clustering and proper assembly of focal adhesion complex . (A) PSAP down-modulation reduced phosphorylation of FAK and paxillin. Cells were incubated in suspension with gentle rotation for 45 min and then plated onto FN- or LN-coated dishes for 45 or 90 min. Whole cell lysates were extracted and equal amount of proteins were used for immunoprecipitation with anti-FAK or-paxillin antibody and immunoblotting with phospho-specific antibodies against Tyr-397, -576, -861, -925 of FAK or Tyr-118 of paxillin. (B) Effect of PSAP down-modulation on β 1A -integrin clustering and focal adhesion complex assembly. Cells were plated onto FN- or LN-coated slides for 2 h, fixed and permeabilized. Immunofluorescence staining was performed with primary antibodies against integrin β 1A , FAK pY397 and paxillin pY118 followed by Cy3 (red) or FITC (green)-conjugated secondary antibodies. F-actin was stained by Oregon Green 488-phalloidin (green). All images were taken by a Leica DM RA2 fluorescence microscope. Consistent data were obtained from three independent experiments.

    Article Snippet: Languino, University of Massachusetts (at 1:100), monoclonal anti-paxillin (Millipore, clone 5H11, 1:1000), and rabbit anti-paxillin-pY118 (Cell signaling, 1:1000).

    Techniques: Activity Assay, Incubation, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy