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TaKaRa n terminal gfp tag
CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies <t>anti-GFP</t> and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged <t>DNMT1</t> protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
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1) Product Images from "Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome"

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0546-4

CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
Figure Legend Snippet: CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

Techniques Used: Western Blot, Purification, Generated, Mutagenesis

2) Product Images from "Regulation of BLM Nucleolar Localization"

Article Title: Regulation of BLM Nucleolar Localization

Journal: Genes

doi: 10.3390/genes7090069

S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p
Figure Legend Snippet: S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p

Techniques Used: Transfection, Staining, Standard Deviation

3) Product Images from "ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility"

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

Journal: Journal of Cell Science

doi: 10.1242/jcs.197434

Src phosphorylation on Tyr376 regulates ARHGAP42 activity. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were transfected to stably express GFP-ARHGAP42-WT versus -Y376F. (A) Expression of ARHGAP42 and Src was confirmed by IB, with actin (bottom) as a control for equal loading. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. (B) Relative RhoA-GTP levels were determined in cell lysates using the G-LISA assay. Values are mean±s.d. RhoA-GTP signal compared to the signal from ARHGAP42-WT-expressing cells, from three independent experiments performed in triplicate. (C) The ability of ARHGAP42 to bind RhoA-CA was analyzed using a GST-RhoA-CA pull-down assay and (D) quantified as a ratio of the amount of indicated GFP-ARHGAP42 variant pulled down with GST-RhoA-CA and the corresponding amount of GFP-ARHGAP42 in the input cell lysate. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.
Figure Legend Snippet: Src phosphorylation on Tyr376 regulates ARHGAP42 activity. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were transfected to stably express GFP-ARHGAP42-WT versus -Y376F. (A) Expression of ARHGAP42 and Src was confirmed by IB, with actin (bottom) as a control for equal loading. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. (B) Relative RhoA-GTP levels were determined in cell lysates using the G-LISA assay. Values are mean±s.d. RhoA-GTP signal compared to the signal from ARHGAP42-WT-expressing cells, from three independent experiments performed in triplicate. (C) The ability of ARHGAP42 to bind RhoA-CA was analyzed using a GST-RhoA-CA pull-down assay and (D) quantified as a ratio of the amount of indicated GFP-ARHGAP42 variant pulled down with GST-RhoA-CA and the corresponding amount of GFP-ARHGAP42 in the input cell lysate. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Techniques Used: Activity Assay, Expressing, Transfection, Stable Transfection, Pull Down Assay, Variant Assay

Src activates the GAP activity of ARHGAP42, requiring the Tyr-376 phosphorylation site. GFP-ARHGAP42 variants, -WT versus -Y376F, were expressed in either nontransformed or v-Src-transformed NIH-3T3 fibroblasts and analyzed 24 h after transfection. (A) Expression and tyrosine phosphorylation of GFP-ARHGAP42 variants was assessed by IP using anti-GFP antibody, and IB with ARHGAP42 antibody (top panel) or anti-pTyr antibody (middle panel). Src activity is indicated by IB of whole cell lysates with antibody against the Src autophosphorylation site (bottom panel). (B) Quantitative analysis of the arborized morphology characteristic of RhoA inhibition in NIH-3T3 cells. Values are mean±s.d. from five independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (C) Subcellular localization of GFP-ARHGAP42 variants and cellular morphology were assessed by fluorescence microscopy of fixed cells. Representative nontransformed cells (left two panels) and v-Src-transformed cells (right two panels) are shown. The podosomal-like structures in the central region of a GFP-ARHGAP42-Y376F-expressing cell are shown in the inset. Scale bars: 30 μm.
Figure Legend Snippet: Src activates the GAP activity of ARHGAP42, requiring the Tyr-376 phosphorylation site. GFP-ARHGAP42 variants, -WT versus -Y376F, were expressed in either nontransformed or v-Src-transformed NIH-3T3 fibroblasts and analyzed 24 h after transfection. (A) Expression and tyrosine phosphorylation of GFP-ARHGAP42 variants was assessed by IP using anti-GFP antibody, and IB with ARHGAP42 antibody (top panel) or anti-pTyr antibody (middle panel). Src activity is indicated by IB of whole cell lysates with antibody against the Src autophosphorylation site (bottom panel). (B) Quantitative analysis of the arborized morphology characteristic of RhoA inhibition in NIH-3T3 cells. Values are mean±s.d. from five independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (C) Subcellular localization of GFP-ARHGAP42 variants and cellular morphology were assessed by fluorescence microscopy of fixed cells. Representative nontransformed cells (left two panels) and v-Src-transformed cells (right two panels) are shown. The podosomal-like structures in the central region of a GFP-ARHGAP42-Y376F-expressing cell are shown in the inset. Scale bars: 30 μm.

Techniques Used: Activity Assay, Transformation Assay, Transfection, Expressing, Inhibition, Fluorescence, Microscopy

Expression of ARHGAP42-ΔGAP increases focal adhesion size; expression of ARHGAP42-ΔBAR promotes focal adhesion turnover and cellular motility. (A) Quantification of focal adhesion size. MEFs expressing ARHGAP42 variants (WT, ΔBAR and ΔGAP) were grown on fibronectin-coated cover slips, fixed, immunostained with an antibody against paxillin, and focal adhesion size was analyzed by fluorescence microscopy. The box-and-whisker plot shows the range of size of focal adhesions. Center line shows the median, box limits indicate the first and third quartiles, whiskers extend to the minimum and maximum values. (B,C) MEFs were cotransfected with GFP-ARHGAP42 expression plasmids (WT, ΔBAR, ΔGAP) and (B) mCherry-zyxin or (C) mCherry-vinculin to mark focal adhesions. After transfection, cells were plated on fibronectin-coated glass bottom dishes and analyzed 48 h later by confocal live-cell microscopy. (B) The percentage of focal adhesions that either assembled or disassembled during a 20 min time interval. (C) FRAP analysis of mCherry-vinculin dynamics in focal adhesions, showing mean recovery halftimes. (A-C) The numbers in the histogram bars indicate the number of focal adhesions analyzed. (D) MEFs stably expressing GFP-ARHGAP42 variants (WT, ΔBAR, ΔGAP) were allowed to migrate for 24 h on a Petri dish and the healed area was subsequently determined by light microscopy followed by analysis using ImageJ software. Values are mean±s.d. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.
Figure Legend Snippet: Expression of ARHGAP42-ΔGAP increases focal adhesion size; expression of ARHGAP42-ΔBAR promotes focal adhesion turnover and cellular motility. (A) Quantification of focal adhesion size. MEFs expressing ARHGAP42 variants (WT, ΔBAR and ΔGAP) were grown on fibronectin-coated cover slips, fixed, immunostained with an antibody against paxillin, and focal adhesion size was analyzed by fluorescence microscopy. The box-and-whisker plot shows the range of size of focal adhesions. Center line shows the median, box limits indicate the first and third quartiles, whiskers extend to the minimum and maximum values. (B,C) MEFs were cotransfected with GFP-ARHGAP42 expression plasmids (WT, ΔBAR, ΔGAP) and (B) mCherry-zyxin or (C) mCherry-vinculin to mark focal adhesions. After transfection, cells were plated on fibronectin-coated glass bottom dishes and analyzed 48 h later by confocal live-cell microscopy. (B) The percentage of focal adhesions that either assembled or disassembled during a 20 min time interval. (C) FRAP analysis of mCherry-vinculin dynamics in focal adhesions, showing mean recovery halftimes. (A-C) The numbers in the histogram bars indicate the number of focal adhesions analyzed. (D) MEFs stably expressing GFP-ARHGAP42 variants (WT, ΔBAR, ΔGAP) were allowed to migrate for 24 h on a Petri dish and the healed area was subsequently determined by light microscopy followed by analysis using ImageJ software. Values are mean±s.d. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Techniques Used: Expressing, Fluorescence, Microscopy, Whisker Assay, Transfection, Stable Transfection, Light Microscopy, Software

Src phosphorylation of ARHGAP42 on Tyr376 regulates focal adhesion size and dynamics of lamellipodia and focal adhesions. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were cotransfected with GFP-ARHGAP42 expression plasmids (WT, Y376F and ΔBAR), and plated on fibronectin-covered glass bottom dishes. At 24 h, cells showing similar levels of GFP-ARHGAP42 fluorescence, judged using an integrated intensity value of the GFP signal per cell and acquired with same settings (exposure, laser power, detector gain, etc.) of the microscope, were analyzed by confocal cell microscopy. (A) Quantitative analysis of focal adhesion size. The box-and-whisker plot shows the range in size of focal adhesions marked by paxillin staining in fixed cells. (B) Quantitative analysis of focal adhesion dynamics in live cells. Values are mean±s.e.m. percentage of dynamic focal adhesions during a 10 min time interval. (A,B) The numbers in the indicate the number of focal adhesions analyzed. (C) Quantitative analysis of lamellipodia velocities. Values are mean±s.e.m. velocities of protruding lamellipodia. The numbers in the histogram bars indicate the number of cells analyzed. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.
Figure Legend Snippet: Src phosphorylation of ARHGAP42 on Tyr376 regulates focal adhesion size and dynamics of lamellipodia and focal adhesions. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were cotransfected with GFP-ARHGAP42 expression plasmids (WT, Y376F and ΔBAR), and plated on fibronectin-covered glass bottom dishes. At 24 h, cells showing similar levels of GFP-ARHGAP42 fluorescence, judged using an integrated intensity value of the GFP signal per cell and acquired with same settings (exposure, laser power, detector gain, etc.) of the microscope, were analyzed by confocal cell microscopy. (A) Quantitative analysis of focal adhesion size. The box-and-whisker plot shows the range in size of focal adhesions marked by paxillin staining in fixed cells. (B) Quantitative analysis of focal adhesion dynamics in live cells. Values are mean±s.e.m. percentage of dynamic focal adhesions during a 10 min time interval. (A,B) The numbers in the indicate the number of focal adhesions analyzed. (C) Quantitative analysis of lamellipodia velocities. Values are mean±s.e.m. velocities of protruding lamellipodia. The numbers in the histogram bars indicate the number of cells analyzed. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Techniques Used: Expressing, Fluorescence, Microscopy, Whisker Assay, Staining

ARHGAP42 with deleted BAR domain has enhanced RhoGAP activity. (A) MEFs were transfected with plasmids expressing GFP-ARHGAP42 variants WT, ΔBAR or ΔGAP, and 24 h later the cell lysates were analyzed by immunoblotting with an antibody raised against mouse ARHGAP42. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. Actin was detected as an additional loading control (bottom panel). The numbers indicate the positions of molecular size markers (kDa). (B) Example of a highly arborized MEF cell expressing GFP-ARHGAP42-ΔBAR. 24 h after transfection, the cell was fixed and visualized for GFP fluorescence. Scale bar: 30 µm. (C) Quantification of arborized morphology in MEFs expressing GFP-ARHGAP42 variants. Values are mean±s.d. from four independent transfections, with 500 fluorescent cells scored per transfection. (D) Deletion of the BAR domain significantly enhances the RhoGAP activity of ARHGAP42. Lysates from MEFs stably expressing ARHGAP42 variants were analyzed using a G-LISA assay to detect GTP-bound RhoA. Values are mean±s.d. RhoGTP signal compared to the signal from ARHGAP42-WT cells from three independent experiments. P -values indicate statistical significance determined by one-way ANOVA followed by Tukey's multiple comparisons test.
Figure Legend Snippet: ARHGAP42 with deleted BAR domain has enhanced RhoGAP activity. (A) MEFs were transfected with plasmids expressing GFP-ARHGAP42 variants WT, ΔBAR or ΔGAP, and 24 h later the cell lysates were analyzed by immunoblotting with an antibody raised against mouse ARHGAP42. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. Actin was detected as an additional loading control (bottom panel). The numbers indicate the positions of molecular size markers (kDa). (B) Example of a highly arborized MEF cell expressing GFP-ARHGAP42-ΔBAR. 24 h after transfection, the cell was fixed and visualized for GFP fluorescence. Scale bar: 30 µm. (C) Quantification of arborized morphology in MEFs expressing GFP-ARHGAP42 variants. Values are mean±s.d. from four independent transfections, with 500 fluorescent cells scored per transfection. (D) Deletion of the BAR domain significantly enhances the RhoGAP activity of ARHGAP42. Lysates from MEFs stably expressing ARHGAP42 variants were analyzed using a G-LISA assay to detect GTP-bound RhoA. Values are mean±s.d. RhoGTP signal compared to the signal from ARHGAP42-WT cells from three independent experiments. P -values indicate statistical significance determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Techniques Used: Activity Assay, Transfection, Expressing, Fluorescence, Stable Transfection

Src promotes phosphorylation of ARHGAP42 Tyr-376. (A,B) Immunoblot analysis of GFP-ARHGAP42 phosphorylation. GFP-ARHGAP42 variants were transiently expressed in (A) COS-7 cells or (B) MEFs, either with or without constitutively active Src-F529, and ARHGAP42 tyrosine phosphorylation was assessed by immunoprecipitation (IP) with anti-GFP antibody followed by immunoblotting (IB) with general anti-pTyr (pTyr) or anti-pTyr376 (pY376) antibody. (B) Prior to immunoprecipitation in MEFs, Src activity was further manipulated by incubating the cells for 2 h in the presence or absence of 5 µM saracatinib. Src-F529 expression was confirmed by immunoblot analysis of total cell lysates with antibody against the Src autophosphorylation site (pSrc). (C) In vitro kinase assay. GFP-ARHGAP42 variants were individually expressed in MEFs and immunoprecipitated using anti-GFP antibody, eluted, and incubated with immunoprecipitated Src-F529. After the kinase reaction had been carried out for 1.5 h, levels of GFP-ARHGAP42 phosphorylation were assessed using a pY376 antibody. The numbers on the right indicate the positions of molecular size markers (kDa).
Figure Legend Snippet: Src promotes phosphorylation of ARHGAP42 Tyr-376. (A,B) Immunoblot analysis of GFP-ARHGAP42 phosphorylation. GFP-ARHGAP42 variants were transiently expressed in (A) COS-7 cells or (B) MEFs, either with or without constitutively active Src-F529, and ARHGAP42 tyrosine phosphorylation was assessed by immunoprecipitation (IP) with anti-GFP antibody followed by immunoblotting (IB) with general anti-pTyr (pTyr) or anti-pTyr376 (pY376) antibody. (B) Prior to immunoprecipitation in MEFs, Src activity was further manipulated by incubating the cells for 2 h in the presence or absence of 5 µM saracatinib. Src-F529 expression was confirmed by immunoblot analysis of total cell lysates with antibody against the Src autophosphorylation site (pSrc). (C) In vitro kinase assay. GFP-ARHGAP42 variants were individually expressed in MEFs and immunoprecipitated using anti-GFP antibody, eluted, and incubated with immunoprecipitated Src-F529. After the kinase reaction had been carried out for 1.5 h, levels of GFP-ARHGAP42 phosphorylation were assessed using a pY376 antibody. The numbers on the right indicate the positions of molecular size markers (kDa).

Techniques Used: Immunoprecipitation, Activity Assay, Expressing, In Vitro, Kinase Assay, Incubation

Domain organization, phylogeny, substrate specificity and subcellular localization of ARHGAP42. (A) Domain organization of ARHGAP42 in comparison to the three other mammalian members of the BAR-PH RhoGAP family. For ARHGAP42, the position of the major site of Src-mediated phosphorylation, Tyr-376, is indicated. OPHN1, oligophrenin-1. (B) Phylogram showing evolutionary relationships among the mammalian BAR-PH RhoGAP family members and to more distant relatives predicted from C. elegans ( Ce T04C9.1A) and Drosophila ( Dm ). (C) ARHGAP42 is a GAP for RhoA and Cdc42, but not Rac1. The GAP domain of ARHGAP42 was bacterially expressed, recovered as a GST fusion protein, and assessed for its activity toward the Rho GTPases RhoA, Rac1 and Cdc42 by measuring the amount of phosphate released by GTP hydrolysis using an in vitro assay. Ras was included as a negative control. Values are mean±s.d. from triplicate assays. (D,E) MEFs were transfected with GFP-ARHGAP42 expression plasmid and viewed 24 h later by fluorescence microscopy of fixed cells. The cells were either immunostained with an antibody against paxillin to mark focal adhesions (D, red) or with phalloidin to mark F-actin (E, red). In the representative cell shown in D, GFP-ARHGAP42 is most prominently localized at the focal adhesions and actin stress fibers. In the representative cell shown in E, GFP-ARHGAP42 is more prominently observed in association with actin stress fibers, as well as in apparent focal adhesions. Scale bar: 30 μm.
Figure Legend Snippet: Domain organization, phylogeny, substrate specificity and subcellular localization of ARHGAP42. (A) Domain organization of ARHGAP42 in comparison to the three other mammalian members of the BAR-PH RhoGAP family. For ARHGAP42, the position of the major site of Src-mediated phosphorylation, Tyr-376, is indicated. OPHN1, oligophrenin-1. (B) Phylogram showing evolutionary relationships among the mammalian BAR-PH RhoGAP family members and to more distant relatives predicted from C. elegans ( Ce T04C9.1A) and Drosophila ( Dm ). (C) ARHGAP42 is a GAP for RhoA and Cdc42, but not Rac1. The GAP domain of ARHGAP42 was bacterially expressed, recovered as a GST fusion protein, and assessed for its activity toward the Rho GTPases RhoA, Rac1 and Cdc42 by measuring the amount of phosphate released by GTP hydrolysis using an in vitro assay. Ras was included as a negative control. Values are mean±s.d. from triplicate assays. (D,E) MEFs were transfected with GFP-ARHGAP42 expression plasmid and viewed 24 h later by fluorescence microscopy of fixed cells. The cells were either immunostained with an antibody against paxillin to mark focal adhesions (D, red) or with phalloidin to mark F-actin (E, red). In the representative cell shown in D, GFP-ARHGAP42 is most prominently localized at the focal adhesions and actin stress fibers. In the representative cell shown in E, GFP-ARHGAP42 is more prominently observed in association with actin stress fibers, as well as in apparent focal adhesions. Scale bar: 30 μm.

Techniques Used: Activity Assay, In Vitro, Negative Control, Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy

Expression of ARHGAP42 promotes membrane tubulation that is enhanced by deletion of the GAP domain. Plasmids expressing GFP-ARHGAP42-WT versus -ΔGAP (or the empty vector) were transfected into COS-7 cells and the cells were analyzed 48 h later. (A) Immunoblot analysis of whole cell lysates shows expression levels of the ARHGAP42 variants, with actin as a control for equal loading. (B) Representative cells expressing GFP-ARHGAP42-WT (left) and GFP-ARHGAP42-ΔGAP (middle and right, exhibiting membrane tabulation). The cells were fixed and visualized for GFP fluorescence. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars: 30 μm. (C) Quantitative analysis of membrane tubulation induced by ARHGAP42 variants. Values are mean±s.d. from four independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.
Figure Legend Snippet: Expression of ARHGAP42 promotes membrane tubulation that is enhanced by deletion of the GAP domain. Plasmids expressing GFP-ARHGAP42-WT versus -ΔGAP (or the empty vector) were transfected into COS-7 cells and the cells were analyzed 48 h later. (A) Immunoblot analysis of whole cell lysates shows expression levels of the ARHGAP42 variants, with actin as a control for equal loading. (B) Representative cells expressing GFP-ARHGAP42-WT (left) and GFP-ARHGAP42-ΔGAP (middle and right, exhibiting membrane tabulation). The cells were fixed and visualized for GFP fluorescence. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars: 30 μm. (C) Quantitative analysis of membrane tubulation induced by ARHGAP42 variants. Values are mean±s.d. from four independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Techniques Used: Expressing, Plasmid Preparation, Transfection, Fluorescence

4) Product Images from "S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *"

Article Title: S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.135095

S100P affects IQGAP1-B-Raf complex formation and reduces EGF-dependent MEK1/2 activation. A , HeLa cells expressing GFP-S100Pwt, GFP-S100PΔ21–25, or YFP-S100A10 were serum-starved and stimulated with 50 ng/ml EGF for 5 min. Cells were lysed,
Figure Legend Snippet: S100P affects IQGAP1-B-Raf complex formation and reduces EGF-dependent MEK1/2 activation. A , HeLa cells expressing GFP-S100Pwt, GFP-S100PΔ21–25, or YFP-S100A10 were serum-starved and stimulated with 50 ng/ml EGF for 5 min. Cells were lysed,

Techniques Used: Activation Assay, Expressing

5) Product Images from "Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome"

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0546-4

CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
Figure Legend Snippet: CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

Techniques Used: Western Blot, Purification, Generated, Mutagenesis

6) Product Images from "A mammalianized synthetic nitroreductase gene for high-level expression"

Article Title: A mammalianized synthetic nitroreductase gene for high-level expression

Journal: BMC Cancer

doi: 10.1186/1471-2407-9-301

The expression of the bacterial nitroreductase gene in COS-7 cells results in protein aggregation . (A) Schematic representation of the coding regions concerning GNTR expression in pGNTR and pGNTRo constructs, respectively. The wild type ntr gene and the codon-optimized ntro gene were N-terminally fused to gfp providing a marker for NTR expression. The bacterial ntr sequence contains several critical codons (red bars) for the expression in mammalian cells. In pGNTRo all critical ntr codons have been adapted to the preferred codon usage in mouse to ensure optimal expression in mammalian cells. (B) Perinuclear structures (white arrowheads) are present in approximately 10% of pGNTR-transfected COS-7 cells, indicating protein aggregation 48 hours after transfection. Protein aggregation was not present in gntro -expressing cells after codon optimization. GFP-labeled nitroreductase (green) and DAPI-stained DNA (blue) are shown in overlay. Scale bar = 50 μm.
Figure Legend Snippet: The expression of the bacterial nitroreductase gene in COS-7 cells results in protein aggregation . (A) Schematic representation of the coding regions concerning GNTR expression in pGNTR and pGNTRo constructs, respectively. The wild type ntr gene and the codon-optimized ntro gene were N-terminally fused to gfp providing a marker for NTR expression. The bacterial ntr sequence contains several critical codons (red bars) for the expression in mammalian cells. In pGNTRo all critical ntr codons have been adapted to the preferred codon usage in mouse to ensure optimal expression in mammalian cells. (B) Perinuclear structures (white arrowheads) are present in approximately 10% of pGNTR-transfected COS-7 cells, indicating protein aggregation 48 hours after transfection. Protein aggregation was not present in gntro -expressing cells after codon optimization. GFP-labeled nitroreductase (green) and DAPI-stained DNA (blue) are shown in overlay. Scale bar = 50 μm.

Techniques Used: Expressing, Construct, Marker, Sequencing, Transfection, Labeling, Staining

The nitroreductase expression in COS-7 cells is improved by codon usage optimization . (A) At the indicated time points cleared cellular lysates (20 μg) of pGNTR and pGNTRo-transfected cells were analyzed by immunoblotting using anti-GFP and anti-actin (loading control) antibodies. (B) Densitometric quantification of GNTR protein levels normalized to actin. Combined data of two independent experiments are represented as means ± SEM. *: p
Figure Legend Snippet: The nitroreductase expression in COS-7 cells is improved by codon usage optimization . (A) At the indicated time points cleared cellular lysates (20 μg) of pGNTR and pGNTRo-transfected cells were analyzed by immunoblotting using anti-GFP and anti-actin (loading control) antibodies. (B) Densitometric quantification of GNTR protein levels normalized to actin. Combined data of two independent experiments are represented as means ± SEM. *: p

Techniques Used: Expressing, Transfection

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Article Title: A mammalianized synthetic nitroreductase gene for high-level expression
Article Snippet: .. To express nitroreductase with a N-terminal GFP-tag both genes were cut out with Eco R I from the respective source vectors and cloned into pEGFP-C1 (Clontech) using Eco R I restriction sites. .. The obtained constructs pGNTR and pGNTRo slightly differed in linker sequence (5 of 17 amino acids) and were used for all cell culture experiments.

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Subcloning:

Article Title: Multiple Cell Adhesion Molecules Shaping a Complex Nicotinic Synapse on Neurons
Article Snippet: .. All full-length or truncated constructs were prepared by subcloning the appropriate sequences into mammalian expression vectors providing either a C- or N-terminal GFP tag (pEGFPN1 or C1 vectors respectively, Clontech, Palo Alto, CA). .. The L1Cyt/NL54 construct was generated by subcloning the entire L1Cyt-GFP cassette, including the intact pCMV promoter and SV40 polyA signal, upstream of the similar NL54-GFP cassette.

Construct:

Article Title: Multiple Cell Adhesion Molecules Shaping a Complex Nicotinic Synapse on Neurons
Article Snippet: .. All full-length or truncated constructs were prepared by subcloning the appropriate sequences into mammalian expression vectors providing either a C- or N-terminal GFP tag (pEGFPN1 or C1 vectors respectively, Clontech, Palo Alto, CA). .. The L1Cyt/NL54 construct was generated by subcloning the entire L1Cyt-GFP cassette, including the intact pCMV promoter and SV40 polyA signal, upstream of the similar NL54-GFP cassette.

Generated:

Article Title: Regulation of BLM Nucleolar Localization
Article Snippet: .. Inserts were transferred into the pEGFP-BLM vector, originally generated by cloning BLM cDNA into a mammalian expression vector containing an N-terminal GFP tag, pEGFP-C1 (Clontech, Mountain View, CA, USA). .. The pYES-BLM mutants and pEGFP-BLM vector were digested with AflII and AsiSI at 37 °C for 1 h, and the remaining steps were carried out as previously described.

Expressing:

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility
Article Snippet: .. For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F. .. Standard molecular methods were employed to introduce the individual deletions or point mutation.

Article Title: Multiple Cell Adhesion Molecules Shaping a Complex Nicotinic Synapse on Neurons
Article Snippet: .. All full-length or truncated constructs were prepared by subcloning the appropriate sequences into mammalian expression vectors providing either a C- or N-terminal GFP tag (pEGFPN1 or C1 vectors respectively, Clontech, Palo Alto, CA). .. The L1Cyt/NL54 construct was generated by subcloning the entire L1Cyt-GFP cassette, including the intact pCMV promoter and SV40 polyA signal, upstream of the similar NL54-GFP cassette.

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: Regulation of BLM Nucleolar Localization
Article Snippet: .. Inserts were transferred into the pEGFP-BLM vector, originally generated by cloning BLM cDNA into a mammalian expression vector containing an N-terminal GFP tag, pEGFP-C1 (Clontech, Mountain View, CA, USA). .. The pYES-BLM mutants and pEGFP-BLM vector were digested with AflII and AsiSI at 37 °C for 1 h, and the remaining steps were carried out as previously described.

Article Title: S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *
Article Snippet: .. For the expression of S100PΔ21–25 fused to an N-terminal GFP tag, the coding sequence of S100PΔ21–25 was cloned into the pEGFP-C2 vector using EcoRI and SalI restriction sites (Clontech). .. Expression of full-length GST-tagged IQGAP1 in SF9 insect cells employed Autographa californica nuclear polyhedrosis baculoviruses encoding GST-IQGAP1.

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Sequencing:

Article Title: S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *
Article Snippet: .. For the expression of S100PΔ21–25 fused to an N-terminal GFP tag, the coding sequence of S100PΔ21–25 was cloned into the pEGFP-C2 vector using EcoRI and SalI restriction sites (Clontech). .. Expression of full-length GST-tagged IQGAP1 in SF9 insect cells employed Autographa californica nuclear polyhedrosis baculoviruses encoding GST-IQGAP1.

Plasmid Preparation:

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility
Article Snippet: .. For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F. .. Standard molecular methods were employed to introduce the individual deletions or point mutation.

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

Article Title: Palmitoylation of human FasL modulates its cell death-inducing function
Article Snippet: .. To produce the N-terminal GFP-tag, FasL cDNA was cloned into the pEGFPC1 vector from Clontech (Mountain View, CA, USA) resulting in pCR33 GFP-FasL . .. The GFP-hFasLC82S and GFP-hFasLA247E constructs were obtained using the Quikchange site-directed mutagenesis kit, with pCR33 GFP-FasL as a template.

Article Title: Regulation of BLM Nucleolar Localization
Article Snippet: .. Inserts were transferred into the pEGFP-BLM vector, originally generated by cloning BLM cDNA into a mammalian expression vector containing an N-terminal GFP tag, pEGFP-C1 (Clontech, Mountain View, CA, USA). .. The pYES-BLM mutants and pEGFP-BLM vector were digested with AflII and AsiSI at 37 °C for 1 h, and the remaining steps were carried out as previously described.

Article Title: S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *
Article Snippet: .. For the expression of S100PΔ21–25 fused to an N-terminal GFP tag, the coding sequence of S100PΔ21–25 was cloned into the pEGFP-C2 vector using EcoRI and SalI restriction sites (Clontech). .. Expression of full-length GST-tagged IQGAP1 in SF9 insect cells employed Autographa californica nuclear polyhedrosis baculoviruses encoding GST-IQGAP1.

Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome
Article Snippet: .. The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich). .. The pEGFP-C1-DNMT1 construct was transfected into competent DH5-alpha cells, and construct fidelity was assessed by sequencing plasmid DNA using primers designed to DNMT1 exons as described in the supplementary information (cDNMT1 sequencing primers).

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    TaKaRa n terminal gfp tag
    CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies <t>anti-GFP</t> and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged <t>DNMT1</t> protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein
    N Terminal Gfp Tag, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

    Journal: Clinical Epigenetics

    Article Title: Genetic variation affecting DNA methylation and the human imprinting disorder, Beckwith-Wiedemann syndrome

    doi: 10.1186/s13148-018-0546-4

    Figure Lengend Snippet: CyDye fluorescent Western blots. a HeLa cell protein lysates present in unpurified and unbound lysate fractions probed with primary antibodies anti-GFP and anti-GAPDH and with secondary antibodies goat-anti-rabbit IgG Cy5 and goat-anti-mouse IgG Cy3. b Purified GFP-tagged DNMT1 protein (DNMT1 225 kDa) and GFP (27 KDa) and DNMT1 variants generated by site-directed mutagenesis. The GFP bands in the fusion protein lanes in ( b ) likely represent cleavage products of the purified fusion protein

    Article Snippet: Generation of DNMT1 plasmids The mammalian expression vector containing the long isoform of wild-type human DNMT1 with N-terminal GFP tag in pEGFP-C1 plasmid (Clontech) was obtained from Prof. Heinrich Leonhardt (Ludwig-Maximilians-University Biocentre, Munich).

    Techniques: Western Blot, Purification, Generated, Mutagenesis

    S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p

    Journal: Genes

    Article Title: Regulation of BLM Nucleolar Localization

    doi: 10.3390/genes7090069

    Figure Lengend Snippet: S1342 and S1345 determine nucleolar localization of BLM. ( A ) Cellular localization of BLM phospho-dead (BLM-S1342A/S1345A) and phospho-mimetic (BLM-S1342D/S1345D) mutants. GM08505 BLM −/− cells were plated on sterile coverslips and transfected with the indicated GFP-tagged BLM plasmids, BLM-WT , BLM-D795A (helicase dead), BLM-S1342A/S1345A and BLM-S1342D/S1345D , using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde 24-h post-transfection, permeabilized with 0.25% Triton-X-100 and blocked with 10% normal goat serum. Nucleoli were stained with anti-nucleophosmin (α-NPM) and Alexa-Fluor fluorescent secondary antibodies and coverslips were mounted with VectaShield plus DAPI mounting medium. ( B ) Quantification of nucleolar localization. Nucleolar localization for 100 cells from 4 to 5 blinded experiments was expressed as percent nucleolar localization. Error bars depict standard deviation. Results were compared to BLM-WT using a Student’s t -test (*** p

    Article Snippet: Inserts were transferred into the pEGFP-BLM vector, originally generated by cloning BLM cDNA into a mammalian expression vector containing an N-terminal GFP tag, pEGFP-C1 (Clontech, Mountain View, CA, USA).

    Techniques: Transfection, Staining, Standard Deviation

    Src phosphorylation on Tyr376 regulates ARHGAP42 activity. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were transfected to stably express GFP-ARHGAP42-WT versus -Y376F. (A) Expression of ARHGAP42 and Src was confirmed by IB, with actin (bottom) as a control for equal loading. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. (B) Relative RhoA-GTP levels were determined in cell lysates using the G-LISA assay. Values are mean±s.d. RhoA-GTP signal compared to the signal from ARHGAP42-WT-expressing cells, from three independent experiments performed in triplicate. (C) The ability of ARHGAP42 to bind RhoA-CA was analyzed using a GST-RhoA-CA pull-down assay and (D) quantified as a ratio of the amount of indicated GFP-ARHGAP42 variant pulled down with GST-RhoA-CA and the corresponding amount of GFP-ARHGAP42 in the input cell lysate. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Src phosphorylation on Tyr376 regulates ARHGAP42 activity. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were transfected to stably express GFP-ARHGAP42-WT versus -Y376F. (A) Expression of ARHGAP42 and Src was confirmed by IB, with actin (bottom) as a control for equal loading. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. (B) Relative RhoA-GTP levels were determined in cell lysates using the G-LISA assay. Values are mean±s.d. RhoA-GTP signal compared to the signal from ARHGAP42-WT-expressing cells, from three independent experiments performed in triplicate. (C) The ability of ARHGAP42 to bind RhoA-CA was analyzed using a GST-RhoA-CA pull-down assay and (D) quantified as a ratio of the amount of indicated GFP-ARHGAP42 variant pulled down with GST-RhoA-CA and the corresponding amount of GFP-ARHGAP42 in the input cell lysate. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Activity Assay, Expressing, Transfection, Stable Transfection, Pull Down Assay, Variant Assay

    Src activates the GAP activity of ARHGAP42, requiring the Tyr-376 phosphorylation site. GFP-ARHGAP42 variants, -WT versus -Y376F, were expressed in either nontransformed or v-Src-transformed NIH-3T3 fibroblasts and analyzed 24 h after transfection. (A) Expression and tyrosine phosphorylation of GFP-ARHGAP42 variants was assessed by IP using anti-GFP antibody, and IB with ARHGAP42 antibody (top panel) or anti-pTyr antibody (middle panel). Src activity is indicated by IB of whole cell lysates with antibody against the Src autophosphorylation site (bottom panel). (B) Quantitative analysis of the arborized morphology characteristic of RhoA inhibition in NIH-3T3 cells. Values are mean±s.d. from five independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (C) Subcellular localization of GFP-ARHGAP42 variants and cellular morphology were assessed by fluorescence microscopy of fixed cells. Representative nontransformed cells (left two panels) and v-Src-transformed cells (right two panels) are shown. The podosomal-like structures in the central region of a GFP-ARHGAP42-Y376F-expressing cell are shown in the inset. Scale bars: 30 μm.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Src activates the GAP activity of ARHGAP42, requiring the Tyr-376 phosphorylation site. GFP-ARHGAP42 variants, -WT versus -Y376F, were expressed in either nontransformed or v-Src-transformed NIH-3T3 fibroblasts and analyzed 24 h after transfection. (A) Expression and tyrosine phosphorylation of GFP-ARHGAP42 variants was assessed by IP using anti-GFP antibody, and IB with ARHGAP42 antibody (top panel) or anti-pTyr antibody (middle panel). Src activity is indicated by IB of whole cell lysates with antibody against the Src autophosphorylation site (bottom panel). (B) Quantitative analysis of the arborized morphology characteristic of RhoA inhibition in NIH-3T3 cells. Values are mean±s.d. from five independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (C) Subcellular localization of GFP-ARHGAP42 variants and cellular morphology were assessed by fluorescence microscopy of fixed cells. Representative nontransformed cells (left two panels) and v-Src-transformed cells (right two panels) are shown. The podosomal-like structures in the central region of a GFP-ARHGAP42-Y376F-expressing cell are shown in the inset. Scale bars: 30 μm.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Activity Assay, Transformation Assay, Transfection, Expressing, Inhibition, Fluorescence, Microscopy

    Expression of ARHGAP42-ΔGAP increases focal adhesion size; expression of ARHGAP42-ΔBAR promotes focal adhesion turnover and cellular motility. (A) Quantification of focal adhesion size. MEFs expressing ARHGAP42 variants (WT, ΔBAR and ΔGAP) were grown on fibronectin-coated cover slips, fixed, immunostained with an antibody against paxillin, and focal adhesion size was analyzed by fluorescence microscopy. The box-and-whisker plot shows the range of size of focal adhesions. Center line shows the median, box limits indicate the first and third quartiles, whiskers extend to the minimum and maximum values. (B,C) MEFs were cotransfected with GFP-ARHGAP42 expression plasmids (WT, ΔBAR, ΔGAP) and (B) mCherry-zyxin or (C) mCherry-vinculin to mark focal adhesions. After transfection, cells were plated on fibronectin-coated glass bottom dishes and analyzed 48 h later by confocal live-cell microscopy. (B) The percentage of focal adhesions that either assembled or disassembled during a 20 min time interval. (C) FRAP analysis of mCherry-vinculin dynamics in focal adhesions, showing mean recovery halftimes. (A-C) The numbers in the histogram bars indicate the number of focal adhesions analyzed. (D) MEFs stably expressing GFP-ARHGAP42 variants (WT, ΔBAR, ΔGAP) were allowed to migrate for 24 h on a Petri dish and the healed area was subsequently determined by light microscopy followed by analysis using ImageJ software. Values are mean±s.d. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Expression of ARHGAP42-ΔGAP increases focal adhesion size; expression of ARHGAP42-ΔBAR promotes focal adhesion turnover and cellular motility. (A) Quantification of focal adhesion size. MEFs expressing ARHGAP42 variants (WT, ΔBAR and ΔGAP) were grown on fibronectin-coated cover slips, fixed, immunostained with an antibody against paxillin, and focal adhesion size was analyzed by fluorescence microscopy. The box-and-whisker plot shows the range of size of focal adhesions. Center line shows the median, box limits indicate the first and third quartiles, whiskers extend to the minimum and maximum values. (B,C) MEFs were cotransfected with GFP-ARHGAP42 expression plasmids (WT, ΔBAR, ΔGAP) and (B) mCherry-zyxin or (C) mCherry-vinculin to mark focal adhesions. After transfection, cells were plated on fibronectin-coated glass bottom dishes and analyzed 48 h later by confocal live-cell microscopy. (B) The percentage of focal adhesions that either assembled or disassembled during a 20 min time interval. (C) FRAP analysis of mCherry-vinculin dynamics in focal adhesions, showing mean recovery halftimes. (A-C) The numbers in the histogram bars indicate the number of focal adhesions analyzed. (D) MEFs stably expressing GFP-ARHGAP42 variants (WT, ΔBAR, ΔGAP) were allowed to migrate for 24 h on a Petri dish and the healed area was subsequently determined by light microscopy followed by analysis using ImageJ software. Values are mean±s.d. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Expressing, Fluorescence, Microscopy, Whisker Assay, Transfection, Stable Transfection, Light Microscopy, Software

    Src phosphorylation of ARHGAP42 on Tyr376 regulates focal adhesion size and dynamics of lamellipodia and focal adhesions. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were cotransfected with GFP-ARHGAP42 expression plasmids (WT, Y376F and ΔBAR), and plated on fibronectin-covered glass bottom dishes. At 24 h, cells showing similar levels of GFP-ARHGAP42 fluorescence, judged using an integrated intensity value of the GFP signal per cell and acquired with same settings (exposure, laser power, detector gain, etc.) of the microscope, were analyzed by confocal cell microscopy. (A) Quantitative analysis of focal adhesion size. The box-and-whisker plot shows the range in size of focal adhesions marked by paxillin staining in fixed cells. (B) Quantitative analysis of focal adhesion dynamics in live cells. Values are mean±s.e.m. percentage of dynamic focal adhesions during a 10 min time interval. (A,B) The numbers in the indicate the number of focal adhesions analyzed. (C) Quantitative analysis of lamellipodia velocities. Values are mean±s.e.m. velocities of protruding lamellipodia. The numbers in the histogram bars indicate the number of cells analyzed. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Src phosphorylation of ARHGAP42 on Tyr376 regulates focal adhesion size and dynamics of lamellipodia and focal adhesions. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were cotransfected with GFP-ARHGAP42 expression plasmids (WT, Y376F and ΔBAR), and plated on fibronectin-covered glass bottom dishes. At 24 h, cells showing similar levels of GFP-ARHGAP42 fluorescence, judged using an integrated intensity value of the GFP signal per cell and acquired with same settings (exposure, laser power, detector gain, etc.) of the microscope, were analyzed by confocal cell microscopy. (A) Quantitative analysis of focal adhesion size. The box-and-whisker plot shows the range in size of focal adhesions marked by paxillin staining in fixed cells. (B) Quantitative analysis of focal adhesion dynamics in live cells. Values are mean±s.e.m. percentage of dynamic focal adhesions during a 10 min time interval. (A,B) The numbers in the indicate the number of focal adhesions analyzed. (C) Quantitative analysis of lamellipodia velocities. Values are mean±s.e.m. velocities of protruding lamellipodia. The numbers in the histogram bars indicate the number of cells analyzed. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Expressing, Fluorescence, Microscopy, Whisker Assay, Staining

    ARHGAP42 with deleted BAR domain has enhanced RhoGAP activity. (A) MEFs were transfected with plasmids expressing GFP-ARHGAP42 variants WT, ΔBAR or ΔGAP, and 24 h later the cell lysates were analyzed by immunoblotting with an antibody raised against mouse ARHGAP42. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. Actin was detected as an additional loading control (bottom panel). The numbers indicate the positions of molecular size markers (kDa). (B) Example of a highly arborized MEF cell expressing GFP-ARHGAP42-ΔBAR. 24 h after transfection, the cell was fixed and visualized for GFP fluorescence. Scale bar: 30 µm. (C) Quantification of arborized morphology in MEFs expressing GFP-ARHGAP42 variants. Values are mean±s.d. from four independent transfections, with 500 fluorescent cells scored per transfection. (D) Deletion of the BAR domain significantly enhances the RhoGAP activity of ARHGAP42. Lysates from MEFs stably expressing ARHGAP42 variants were analyzed using a G-LISA assay to detect GTP-bound RhoA. Values are mean±s.d. RhoGTP signal compared to the signal from ARHGAP42-WT cells from three independent experiments. P -values indicate statistical significance determined by one-way ANOVA followed by Tukey's multiple comparisons test.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: ARHGAP42 with deleted BAR domain has enhanced RhoGAP activity. (A) MEFs were transfected with plasmids expressing GFP-ARHGAP42 variants WT, ΔBAR or ΔGAP, and 24 h later the cell lysates were analyzed by immunoblotting with an antibody raised against mouse ARHGAP42. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. Actin was detected as an additional loading control (bottom panel). The numbers indicate the positions of molecular size markers (kDa). (B) Example of a highly arborized MEF cell expressing GFP-ARHGAP42-ΔBAR. 24 h after transfection, the cell was fixed and visualized for GFP fluorescence. Scale bar: 30 µm. (C) Quantification of arborized morphology in MEFs expressing GFP-ARHGAP42 variants. Values are mean±s.d. from four independent transfections, with 500 fluorescent cells scored per transfection. (D) Deletion of the BAR domain significantly enhances the RhoGAP activity of ARHGAP42. Lysates from MEFs stably expressing ARHGAP42 variants were analyzed using a G-LISA assay to detect GTP-bound RhoA. Values are mean±s.d. RhoGTP signal compared to the signal from ARHGAP42-WT cells from three independent experiments. P -values indicate statistical significance determined by one-way ANOVA followed by Tukey's multiple comparisons test.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Activity Assay, Transfection, Expressing, Fluorescence, Stable Transfection

    Src promotes phosphorylation of ARHGAP42 Tyr-376. (A,B) Immunoblot analysis of GFP-ARHGAP42 phosphorylation. GFP-ARHGAP42 variants were transiently expressed in (A) COS-7 cells or (B) MEFs, either with or without constitutively active Src-F529, and ARHGAP42 tyrosine phosphorylation was assessed by immunoprecipitation (IP) with anti-GFP antibody followed by immunoblotting (IB) with general anti-pTyr (pTyr) or anti-pTyr376 (pY376) antibody. (B) Prior to immunoprecipitation in MEFs, Src activity was further manipulated by incubating the cells for 2 h in the presence or absence of 5 µM saracatinib. Src-F529 expression was confirmed by immunoblot analysis of total cell lysates with antibody against the Src autophosphorylation site (pSrc). (C) In vitro kinase assay. GFP-ARHGAP42 variants were individually expressed in MEFs and immunoprecipitated using anti-GFP antibody, eluted, and incubated with immunoprecipitated Src-F529. After the kinase reaction had been carried out for 1.5 h, levels of GFP-ARHGAP42 phosphorylation were assessed using a pY376 antibody. The numbers on the right indicate the positions of molecular size markers (kDa).

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Src promotes phosphorylation of ARHGAP42 Tyr-376. (A,B) Immunoblot analysis of GFP-ARHGAP42 phosphorylation. GFP-ARHGAP42 variants were transiently expressed in (A) COS-7 cells or (B) MEFs, either with or without constitutively active Src-F529, and ARHGAP42 tyrosine phosphorylation was assessed by immunoprecipitation (IP) with anti-GFP antibody followed by immunoblotting (IB) with general anti-pTyr (pTyr) or anti-pTyr376 (pY376) antibody. (B) Prior to immunoprecipitation in MEFs, Src activity was further manipulated by incubating the cells for 2 h in the presence or absence of 5 µM saracatinib. Src-F529 expression was confirmed by immunoblot analysis of total cell lysates with antibody against the Src autophosphorylation site (pSrc). (C) In vitro kinase assay. GFP-ARHGAP42 variants were individually expressed in MEFs and immunoprecipitated using anti-GFP antibody, eluted, and incubated with immunoprecipitated Src-F529. After the kinase reaction had been carried out for 1.5 h, levels of GFP-ARHGAP42 phosphorylation were assessed using a pY376 antibody. The numbers on the right indicate the positions of molecular size markers (kDa).

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Immunoprecipitation, Activity Assay, Expressing, In Vitro, Kinase Assay, Incubation

    Domain organization, phylogeny, substrate specificity and subcellular localization of ARHGAP42. (A) Domain organization of ARHGAP42 in comparison to the three other mammalian members of the BAR-PH RhoGAP family. For ARHGAP42, the position of the major site of Src-mediated phosphorylation, Tyr-376, is indicated. OPHN1, oligophrenin-1. (B) Phylogram showing evolutionary relationships among the mammalian BAR-PH RhoGAP family members and to more distant relatives predicted from C. elegans ( Ce T04C9.1A) and Drosophila ( Dm ). (C) ARHGAP42 is a GAP for RhoA and Cdc42, but not Rac1. The GAP domain of ARHGAP42 was bacterially expressed, recovered as a GST fusion protein, and assessed for its activity toward the Rho GTPases RhoA, Rac1 and Cdc42 by measuring the amount of phosphate released by GTP hydrolysis using an in vitro assay. Ras was included as a negative control. Values are mean±s.d. from triplicate assays. (D,E) MEFs were transfected with GFP-ARHGAP42 expression plasmid and viewed 24 h later by fluorescence microscopy of fixed cells. The cells were either immunostained with an antibody against paxillin to mark focal adhesions (D, red) or with phalloidin to mark F-actin (E, red). In the representative cell shown in D, GFP-ARHGAP42 is most prominently localized at the focal adhesions and actin stress fibers. In the representative cell shown in E, GFP-ARHGAP42 is more prominently observed in association with actin stress fibers, as well as in apparent focal adhesions. Scale bar: 30 μm.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Domain organization, phylogeny, substrate specificity and subcellular localization of ARHGAP42. (A) Domain organization of ARHGAP42 in comparison to the three other mammalian members of the BAR-PH RhoGAP family. For ARHGAP42, the position of the major site of Src-mediated phosphorylation, Tyr-376, is indicated. OPHN1, oligophrenin-1. (B) Phylogram showing evolutionary relationships among the mammalian BAR-PH RhoGAP family members and to more distant relatives predicted from C. elegans ( Ce T04C9.1A) and Drosophila ( Dm ). (C) ARHGAP42 is a GAP for RhoA and Cdc42, but not Rac1. The GAP domain of ARHGAP42 was bacterially expressed, recovered as a GST fusion protein, and assessed for its activity toward the Rho GTPases RhoA, Rac1 and Cdc42 by measuring the amount of phosphate released by GTP hydrolysis using an in vitro assay. Ras was included as a negative control. Values are mean±s.d. from triplicate assays. (D,E) MEFs were transfected with GFP-ARHGAP42 expression plasmid and viewed 24 h later by fluorescence microscopy of fixed cells. The cells were either immunostained with an antibody against paxillin to mark focal adhesions (D, red) or with phalloidin to mark F-actin (E, red). In the representative cell shown in D, GFP-ARHGAP42 is most prominently localized at the focal adhesions and actin stress fibers. In the representative cell shown in E, GFP-ARHGAP42 is more prominently observed in association with actin stress fibers, as well as in apparent focal adhesions. Scale bar: 30 μm.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Activity Assay, In Vitro, Negative Control, Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy

    Expression of ARHGAP42 promotes membrane tubulation that is enhanced by deletion of the GAP domain. Plasmids expressing GFP-ARHGAP42-WT versus -ΔGAP (or the empty vector) were transfected into COS-7 cells and the cells were analyzed 48 h later. (A) Immunoblot analysis of whole cell lysates shows expression levels of the ARHGAP42 variants, with actin as a control for equal loading. (B) Representative cells expressing GFP-ARHGAP42-WT (left) and GFP-ARHGAP42-ΔGAP (middle and right, exhibiting membrane tabulation). The cells were fixed and visualized for GFP fluorescence. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars: 30 μm. (C) Quantitative analysis of membrane tubulation induced by ARHGAP42 variants. Values are mean±s.d. from four independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.

    Journal: Journal of Cell Science

    Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

    doi: 10.1242/jcs.197434

    Figure Lengend Snippet: Expression of ARHGAP42 promotes membrane tubulation that is enhanced by deletion of the GAP domain. Plasmids expressing GFP-ARHGAP42-WT versus -ΔGAP (or the empty vector) were transfected into COS-7 cells and the cells were analyzed 48 h later. (A) Immunoblot analysis of whole cell lysates shows expression levels of the ARHGAP42 variants, with actin as a control for equal loading. (B) Representative cells expressing GFP-ARHGAP42-WT (left) and GFP-ARHGAP42-ΔGAP (middle and right, exhibiting membrane tabulation). The cells were fixed and visualized for GFP fluorescence. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars: 30 μm. (C) Quantitative analysis of membrane tubulation induced by ARHGAP42 variants. Values are mean±s.d. from four independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.

    Article Snippet: For expression of mouse ARHGAP42 variants carrying an N-terminal GFP tag, vector pEGFP-C1 (Clontech) was used in the construction of plasmids pEGFP-C1-mARHGAP42-WT, -ΔBAR, -ΔGAP, -ΔSH3, and -Y376F.

    Techniques: Expressing, Plasmid Preparation, Transfection, Fluorescence

    S100P affects IQGAP1-B-Raf complex formation and reduces EGF-dependent MEK1/2 activation. A , HeLa cells expressing GFP-S100Pwt, GFP-S100PΔ21–25, or YFP-S100A10 were serum-starved and stimulated with 50 ng/ml EGF for 5 min. Cells were lysed,

    Journal: The Journal of Biological Chemistry

    Article Title: S100P Is a Novel Interaction Partner and Regulator of IQGAP1 *

    doi: 10.1074/jbc.M110.135095

    Figure Lengend Snippet: S100P affects IQGAP1-B-Raf complex formation and reduces EGF-dependent MEK1/2 activation. A , HeLa cells expressing GFP-S100Pwt, GFP-S100PΔ21–25, or YFP-S100A10 were serum-starved and stimulated with 50 ng/ml EGF for 5 min. Cells were lysed,

    Article Snippet: For the expression of S100PΔ21–25 fused to an N-terminal GFP tag, the coding sequence of S100PΔ21–25 was cloned into the pEGFP-C2 vector using EcoRI and SalI restriction sites (Clontech).

    Techniques: Activation Assay, Expressing