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pgex-2t knt1 201–445  (GenScript corporation)
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GenScript corporation pgex-2t knt1 201–445
Rab18 directly interacts with <t>KNT1.</t> (a) Representative image of a cell transfected with GFP-Rab18 WT and RFP-vinculin and immunostained with an antibody against KNT1. The insets show regions (white squares) where GFP-Rab18 and KNT1 colocalize in proximity of the plasma membrane with the FA protein vinculin (cyan). Scale bar: 10 µm; insets: 1 µm. (b) U2OS cells were transiently transfected with GFP, GFP-Rab18 WT, GFP-Rab18 Q67L, or GFP-Rab18 S22N, lysed, and subjected to IP with GFP or control magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (c) Schematic representation of KNT1 FL and deletion mutants used in this study. The green square indicates the ER membrane-binding domain of KNT1. (d) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, TM, K1, KNT + , or KNT − , lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (e) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, or Δ201–445, lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (f) Top: Coomassie blue staining of bacterially expressed and purified GST, GST-KNT1 (201–445), and His-Rab18 Q67L. Bottom: Purified GST or GST-KNT1 (201–445) was incubated with purified His-Rab18 Q67L. Samples were subjected to affinity chromatography followed by Western blot analysis using antibodies specific to GST and Rab18.
Pgex 2t Knt1 201–445, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Rab18 directly interacts with KNT1. (a) Representative image of a cell transfected with GFP-Rab18 WT and RFP-vinculin and immunostained with an antibody against KNT1. The insets show regions (white squares) where GFP-Rab18 and KNT1 colocalize in proximity of the plasma membrane with the FA protein vinculin (cyan). Scale bar: 10 µm; insets: 1 µm. (b) U2OS cells were transiently transfected with GFP, GFP-Rab18 WT, GFP-Rab18 Q67L, or GFP-Rab18 S22N, lysed, and subjected to IP with GFP or control magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (c) Schematic representation of KNT1 FL and deletion mutants used in this study. The green square indicates the ER membrane-binding domain of KNT1. (d) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, TM, K1, KNT + , or KNT − , lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (e) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, or Δ201–445, lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (f) Top: Coomassie blue staining of bacterially expressed and purified GST, GST-KNT1 (201–445), and His-Rab18 Q67L. Bottom: Purified GST or GST-KNT1 (201–445) was incubated with purified His-Rab18 Q67L. Samples were subjected to affinity chromatography followed by Western blot analysis using antibodies specific to GST and Rab18.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: Rab18 directly interacts with KNT1. (a) Representative image of a cell transfected with GFP-Rab18 WT and RFP-vinculin and immunostained with an antibody against KNT1. The insets show regions (white squares) where GFP-Rab18 and KNT1 colocalize in proximity of the plasma membrane with the FA protein vinculin (cyan). Scale bar: 10 µm; insets: 1 µm. (b) U2OS cells were transiently transfected with GFP, GFP-Rab18 WT, GFP-Rab18 Q67L, or GFP-Rab18 S22N, lysed, and subjected to IP with GFP or control magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (c) Schematic representation of KNT1 FL and deletion mutants used in this study. The green square indicates the ER membrane-binding domain of KNT1. (d) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, TM, K1, KNT + , or KNT − , lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (e) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, or Δ201–445, lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (f) Top: Coomassie blue staining of bacterially expressed and purified GST, GST-KNT1 (201–445), and His-Rab18 Q67L. Bottom: Purified GST or GST-KNT1 (201–445) was incubated with purified His-Rab18 Q67L. Samples were subjected to affinity chromatography followed by Western blot analysis using antibodies specific to GST and Rab18.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Transfection, Clinical Proteomics, Membrane, Control, Western Blot, Binding Assay, Immunoprecipitation, Staining, Purification, Incubation, Affinity Chromatography

Rab18 binding is required for KNT1 localization at the peripheral tubular ER. (a) U2OS cells transfected with siRNA control or siRNA against Rab18, or depleted for Rab18 and subsequently transfected with GFP-Rab18, were fixed and stained with DAPI and anti-KNT1 antibody. The insets show magnifications of the boxed areas and illustrate examples of KNT1 distribution under the different conditions. The inset shows the GFP channel for the sample transfected with GFP-Rab18. Images represent maximum-intensity projections from Z-stacks. Scale bar: 20 µm; inset: 10 µm. (b) Quantification of KNT1 intracellular distribution in control cells, cells knocked down for Rab18, and cells knocked down for Rab18 and transfected with GFP-Rab18. The graph represents the percentage of perinuclear KNT1 over the total. Data represent the mean ± SEM of three independent experiments ( n > 60). *, P < 0.05; **, P < 0.01. (c) U2OS cells were transfected with control siRNA, siRNA against Rab18, or siRNA depleted for Rab18 and subsequently transfected with GFP-Rab18 or stably transfected with GFP-KNT1 FL or GFP-KNT1 Δ201–445. Cells were fixed and stained with anti-KNT1 and CLIMP-63 or RTN-3 antibody. Left: ER sheets area labeled by CLIMP-63 (top) and ER tubular area labeled by RTN-3 (bottom). Insets: GFP channel for the cells transfected with GFP-Rab18. Scale bar: 10 µm. (d and e) Quantification of KNT1 intracellular distribution in sheet (CLIMP-63; d) and tubule (RTN-3; e) area over the total. Data represent the mean ± SEM of three independent experiments ( n > 60). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: Rab18 binding is required for KNT1 localization at the peripheral tubular ER. (a) U2OS cells transfected with siRNA control or siRNA against Rab18, or depleted for Rab18 and subsequently transfected with GFP-Rab18, were fixed and stained with DAPI and anti-KNT1 antibody. The insets show magnifications of the boxed areas and illustrate examples of KNT1 distribution under the different conditions. The inset shows the GFP channel for the sample transfected with GFP-Rab18. Images represent maximum-intensity projections from Z-stacks. Scale bar: 20 µm; inset: 10 µm. (b) Quantification of KNT1 intracellular distribution in control cells, cells knocked down for Rab18, and cells knocked down for Rab18 and transfected with GFP-Rab18. The graph represents the percentage of perinuclear KNT1 over the total. Data represent the mean ± SEM of three independent experiments ( n > 60). *, P < 0.05; **, P < 0.01. (c) U2OS cells were transfected with control siRNA, siRNA against Rab18, or siRNA depleted for Rab18 and subsequently transfected with GFP-Rab18 or stably transfected with GFP-KNT1 FL or GFP-KNT1 Δ201–445. Cells were fixed and stained with anti-KNT1 and CLIMP-63 or RTN-3 antibody. Left: ER sheets area labeled by CLIMP-63 (top) and ER tubular area labeled by RTN-3 (bottom). Insets: GFP channel for the cells transfected with GFP-Rab18. Scale bar: 10 µm. (d and e) Quantification of KNT1 intracellular distribution in sheet (CLIMP-63; d) and tubule (RTN-3; e) area over the total. Data represent the mean ± SEM of three independent experiments ( n > 60). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Binding Assay, Transfection, Control, Staining, Stable Transfection, Labeling

KNT1 is an adaptor that connects Rab18 to KIF5B. (a) U2OS cells treated with either control siRNA or siRNA directed against KNT1, and transfected with either GFP or GFP-Rab18, were lysed and immunoprecipitated with GFP magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (b) U2OS cells treated with either control siRNA or siRNA directed against Rab18, and transfected with either GFP or KIF5B-GFP, were lysed and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: KNT1 is an adaptor that connects Rab18 to KIF5B. (a) U2OS cells treated with either control siRNA or siRNA directed against KNT1, and transfected with either GFP or GFP-Rab18, were lysed and immunoprecipitated with GFP magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (b) U2OS cells treated with either control siRNA or siRNA directed against Rab18, and transfected with either GFP or KIF5B-GFP, were lysed and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Control, Transfection, Immunoprecipitation, Western Blot

Formation of ER–FA contacts is dependent on the binding of Rab18 and KIF5B to KNT1. (a) U2OS cells silenced with siRNA control or siRNA targeting Rab18 were cotransfected with ER-GFP and RFP-vinculin. The images represent maximum-intensity projections from Z-stacks. The insets show magnifications of the boxed regions. Scale bar: 10 µm; insets: 2 µm. (b) Quantification of the percentage of FAs in contact with the ER. The graph represents the mean ± SEM from three independent experiments ( n > 30 cells). ***, P < 0.001. (c) U2OS cells were treated with either control siRNA or siRNA directed against KNT1 and transfected with either dsRed-ER alone or together with either GFP KNT1 FL, TM, or Δ201–445. Cells were fixed and stained with anti-vinculin antibody. The images represent maximum-intensity projections from Z-stacks. The insets show magnification of boxed regions to illustrate contact points between ER and FAs. Scale bars: 10 µm; insets: 2 µm. (d) Quantification of the percentage of FAs in contact with the ER. The graph represents the mean ± SEM from three independent experiments ( n > 30 cells). *, P < 0.05; ***, P ≤ 0.001; n.s., not significant.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: Formation of ER–FA contacts is dependent on the binding of Rab18 and KIF5B to KNT1. (a) U2OS cells silenced with siRNA control or siRNA targeting Rab18 were cotransfected with ER-GFP and RFP-vinculin. The images represent maximum-intensity projections from Z-stacks. The insets show magnifications of the boxed regions. Scale bar: 10 µm; insets: 2 µm. (b) Quantification of the percentage of FAs in contact with the ER. The graph represents the mean ± SEM from three independent experiments ( n > 30 cells). ***, P < 0.001. (c) U2OS cells were treated with either control siRNA or siRNA directed against KNT1 and transfected with either dsRed-ER alone or together with either GFP KNT1 FL, TM, or Δ201–445. Cells were fixed and stained with anti-vinculin antibody. The images represent maximum-intensity projections from Z-stacks. The insets show magnification of boxed regions to illustrate contact points between ER and FAs. Scale bars: 10 µm; insets: 2 µm. (d) Quantification of the percentage of FAs in contact with the ER. The graph represents the mean ± SEM from three independent experiments ( n > 30 cells). *, P < 0.05; ***, P ≤ 0.001; n.s., not significant.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Binding Assay, Control, Transfection, Staining

Rab18-KNT1 interaction regulates FA size . (a) U2OS cells silenced with siRNA control, siRNA against Rab18, or silenced with siRNA against Rab18 and subsequently transfected with GFP-Rab18 were fixed and stained with Hoechst, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 10 µm. (b–d) Quantification of the size (b) and number of FAs per cell (c) or per 100 µm 2 cell area (d). The graphs represent the mean ± SEM for three independent experiments ( n > 120 cells) *, P < 0.05; **, P < 0.01; ***, P < 0.001. (e) U2OS cells stably transfected with GFP-KNT1 FL, K1, or Δ201–445 were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 10 µm. (f–h) Quantification of the size (f) and number of FAs per cell (g) or per 100 µm 2 cell area (h). The graphs represent the mean ± SEM for three independent experiments ( n > 90 cells). *, P < 0.05.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: Rab18-KNT1 interaction regulates FA size . (a) U2OS cells silenced with siRNA control, siRNA against Rab18, or silenced with siRNA against Rab18 and subsequently transfected with GFP-Rab18 were fixed and stained with Hoechst, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 10 µm. (b–d) Quantification of the size (b) and number of FAs per cell (c) or per 100 µm 2 cell area (d). The graphs represent the mean ± SEM for three independent experiments ( n > 120 cells) *, P < 0.05; **, P < 0.01; ***, P < 0.001. (e) U2OS cells stably transfected with GFP-KNT1 FL, K1, or Δ201–445 were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 10 µm. (f–h) Quantification of the size (f) and number of FAs per cell (g) or per 100 µm 2 cell area (h). The graphs represent the mean ± SEM for three independent experiments ( n > 90 cells). *, P < 0.05.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Control, Transfection, Staining, Stable Transfection

Impaired binding of Rab18 to KNT1 affects FA assembly and disassembly rate. (a) Control or Rab18-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 µm. (b) Rainbow color representation of FA appearance and disappearance over time from cells shown in panel a and for U2OS cells stably expressing GFP-KNT1 K1 or Δ201–445. Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bars: 10 µm; insets: 2 µm. (c) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is expressed as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell ( n > 6), per condition, and per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum

doi: 10.1083/jcb.201809020

Figure Lengend Snippet: Impaired binding of Rab18 to KNT1 affects FA assembly and disassembly rate. (a) Control or Rab18-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 µm. (b) Rainbow color representation of FA appearance and disappearance over time from cells shown in panel a and for U2OS cells stably expressing GFP-KNT1 K1 or Δ201–445. Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bars: 10 µm; insets: 2 µm. (c) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is expressed as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell ( n > 6), per condition, and per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: pEGFP-C1 was purchased from BD Biosciences, Clontech. pEGFP-C1 Rab18 WT, pEGFP-C1 Rab18 Q67L, pEGFP-C1 Rab18 S22N, pcDNA 3.1 HA-Rab18 WT, pEGFP-C1 KNT1 Δ201–445, pGEX-2T KNT1 201–445, pET-16b His Rab18-Q67L, and siRNA-resistant pEGFP-C1 Rab18 containing silent mutations (used in rescue experiments) were purchased from GenScript.

Techniques: Binding Assay, Control, Transfection, Stable Transfection, Expressing