anti ubiquitin antibody  (Millipore)


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    Structured Review

    Millipore anti ubiquitin antibody
    Vpr inhibits ubiquitination of histone H1 variants by CRL4 DCAF1 (A) Schematic of histone h1 protein domains (top; positions indicated are for histone H1.2). Heatmap of phosphorylation (P) and ubiquitination (U) changes on histone H1 variants. All sites are aligned to the sequence positions of histone H1.2. Cells with a red outline were identified by peptides that cannot distinguish between histone variants. (B) Denaturing <t>ubiquitin</t> immunoprecipitation analysis of histone H1.2 with Vpr titration (C) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with MLN4924 titration (D) Schematic of experimental and data analysis workflows for quantitative affinity purification and mass spectrometry analysis of histone H1.2 in the presence and absence of Vpr co-expression (E) Volcano plot of histone H1.2 protein binding changes in response to Vpr expression. Proteins with |log 2 fold-change| > 0.58 (i.e., 1.5-fold change) and adjusted p-value
    Anti Ubiquitin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 16270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 16270 article reviews
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    anti ubiquitin antibody - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling"

    Article Title: Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling

    Journal: bioRxiv

    doi: 10.1101/2020.01.06.896365

    Vpr inhibits ubiquitination of histone H1 variants by CRL4 DCAF1 (A) Schematic of histone h1 protein domains (top; positions indicated are for histone H1.2). Heatmap of phosphorylation (P) and ubiquitination (U) changes on histone H1 variants. All sites are aligned to the sequence positions of histone H1.2. Cells with a red outline were identified by peptides that cannot distinguish between histone variants. (B) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with Vpr titration (C) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with MLN4924 titration (D) Schematic of experimental and data analysis workflows for quantitative affinity purification and mass spectrometry analysis of histone H1.2 in the presence and absence of Vpr co-expression (E) Volcano plot of histone H1.2 protein binding changes in response to Vpr expression. Proteins with |log 2 fold-change| > 0.58 (i.e., 1.5-fold change) and adjusted p-value
    Figure Legend Snippet: Vpr inhibits ubiquitination of histone H1 variants by CRL4 DCAF1 (A) Schematic of histone h1 protein domains (top; positions indicated are for histone H1.2). Heatmap of phosphorylation (P) and ubiquitination (U) changes on histone H1 variants. All sites are aligned to the sequence positions of histone H1.2. Cells with a red outline were identified by peptides that cannot distinguish between histone variants. (B) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with Vpr titration (C) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with MLN4924 titration (D) Schematic of experimental and data analysis workflows for quantitative affinity purification and mass spectrometry analysis of histone H1.2 in the presence and absence of Vpr co-expression (E) Volcano plot of histone H1.2 protein binding changes in response to Vpr expression. Proteins with |log 2 fold-change| > 0.58 (i.e., 1.5-fold change) and adjusted p-value

    Techniques Used: Sequencing, Immunoprecipitation, Titration, Affinity Purification, Mass Spectrometry, Expressing, Protein Binding

    2) Product Images from "FOXK1 interaction with FHL2 promotes proliferation, invasion and metastasis in colorectal cancer"

    Article Title: FOXK1 interaction with FHL2 promotes proliferation, invasion and metastasis in colorectal cancer

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2016.68

    FOXK1 and FHL2 promote the proliferation and EMT in CRC cell. ( a ) Expression levels of FHL2 were detected by western blot analysis in SW480 cells, which were transfected with FOXK1 overexpressing plasmids, followed by transfection with FHL2 siRNA or Scr siRNA as a negative control. ( b ) SW480 stable transfectants of FOXK1, by transfection with FHL2 siRNA or Scr siRNA for 48 h, were subjected to the EdU incorporation assay; **** P
    Figure Legend Snippet: FOXK1 and FHL2 promote the proliferation and EMT in CRC cell. ( a ) Expression levels of FHL2 were detected by western blot analysis in SW480 cells, which were transfected with FOXK1 overexpressing plasmids, followed by transfection with FHL2 siRNA or Scr siRNA as a negative control. ( b ) SW480 stable transfectants of FOXK1, by transfection with FHL2 siRNA or Scr siRNA for 48 h, were subjected to the EdU incorporation assay; **** P

    Techniques Used: Expressing, Western Blot, Transfection, Negative Control

    Interaction between FHL2 and FOXK1 proteins in CRC cells. ( a ) Double staining of FHL2 and FOXK1 in SW480 cells with Hoechst by confocal microscopy. ( b ) Western blotting analysis of FOXK1 and FHL2 expression in the indicated CRC cells. ( c ) PCI-flag-FHL2 plasmid was transfected into SW480 and SW620 cells. Immunoprecipitation was performed with anti-flag antibody, and pre-immune normal mouse immunoglobulin G (nm IgG) was used as control. Western blotting was performed with anti-FOXK1 antibody. The IP blot was probed with indicated antibodies to show the input of whole-cell lysates. IP, immunoprecipitation; Wb, western blot. ( d ) Cell lysates of SW480 and SW620 cells were immmunoprecipitated by anti-FOXK1 antibody or the control antibody, normal rabbit immunoglobulin G (nr IgG). Western blotting was carried out with anti-FHL2 antibody. The IP blot was probed with indicated antibodies to show the input. All these experiments were repeated two to three times with similar findings. Scale bars represent 20 μm in a .
    Figure Legend Snippet: Interaction between FHL2 and FOXK1 proteins in CRC cells. ( a ) Double staining of FHL2 and FOXK1 in SW480 cells with Hoechst by confocal microscopy. ( b ) Western blotting analysis of FOXK1 and FHL2 expression in the indicated CRC cells. ( c ) PCI-flag-FHL2 plasmid was transfected into SW480 and SW620 cells. Immunoprecipitation was performed with anti-flag antibody, and pre-immune normal mouse immunoglobulin G (nm IgG) was used as control. Western blotting was performed with anti-FOXK1 antibody. The IP blot was probed with indicated antibodies to show the input of whole-cell lysates. IP, immunoprecipitation; Wb, western blot. ( d ) Cell lysates of SW480 and SW620 cells were immmunoprecipitated by anti-FOXK1 antibody or the control antibody, normal rabbit immunoglobulin G (nr IgG). Western blotting was carried out with anti-FHL2 antibody. The IP blot was probed with indicated antibodies to show the input. All these experiments were repeated two to three times with similar findings. Scale bars represent 20 μm in a .

    Techniques Used: Double Staining, Confocal Microscopy, Western Blot, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation

    FOXK1 synergizes with FHL2 to promote tumour proliferation and metastasis in vivo. ( a ) Evaluation of tumorigenesis in nude mice subcutaneously injected with SW480-Vector, SW480-FOXK1 and SW480-FOXK1–FHL2-shRNA cells. Images were captured on day 30 after injection. ( b ) Tumour size was measured five days after tumour cell inoculation in each group. ** P
    Figure Legend Snippet: FOXK1 synergizes with FHL2 to promote tumour proliferation and metastasis in vivo. ( a ) Evaluation of tumorigenesis in nude mice subcutaneously injected with SW480-Vector, SW480-FOXK1 and SW480-FOXK1–FHL2-shRNA cells. Images were captured on day 30 after injection. ( b ) Tumour size was measured five days after tumour cell inoculation in each group. ** P

    Techniques Used: In Vivo, Mouse Assay, Injection, Plasmid Preparation, shRNA

    3) Product Images from "Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1"

    Article Title: Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2013.251181

    Bicarbonate permeability of tsA201 cells as a function of intracellular CA activity, A i
    Figure Legend Snippet: Bicarbonate permeability of tsA201 cells as a function of intracellular CA activity, A i

    Techniques Used: Permeability, Activity Assay

    FRET measurements of CyPet- and YPet-labelled fusion proteins in tsA201 cells
    Figure Legend Snippet: FRET measurements of CyPet- and YPet-labelled fusion proteins in tsA201 cells

    Techniques Used:

    Carbonic anhydrase activity and HCO 3 − permeability in native and YPet-mAE1-expressing tsA201 cells
    Figure Legend Snippet: Carbonic anhydrase activity and HCO 3 − permeability in native and YPet-mAE1-expressing tsA201 cells

    Techniques Used: Activity Assay, Permeability, Expressing

    4) Product Images from "HOXB13, a Target of DNMT3B, Is Methylated at an Upstream CpG Island, and Functions as a Tumor Suppressor in Primary Colorectal Tumors"

    Article Title: HOXB13, a Target of DNMT3B, Is Methylated at an Upstream CpG Island, and Functions as a Tumor Suppressor in Primary Colorectal Tumors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010338

    HOXB13 but not TBX18 inhibited ability of RKO cells to form tumor in nude mice. One million cells in PBS (100 µl) were injected into the flanks of nude mice and tumor size was monitored every week using a caliper. After 25 days tumors were excised and their weights were documented. A. Time course of the progression of tumor growth. B. Photograph of the tumors developed by vector-transfected, HoxB13 or TBX18 expressing cells. C. Tumor weight at the time of sacrifice. D. Western blot analysis of ectopic TBX-18 in the tumors T1 to T4 (shown in A) with anti-Flag antibody.
    Figure Legend Snippet: HOXB13 but not TBX18 inhibited ability of RKO cells to form tumor in nude mice. One million cells in PBS (100 µl) were injected into the flanks of nude mice and tumor size was monitored every week using a caliper. After 25 days tumors were excised and their weights were documented. A. Time course of the progression of tumor growth. B. Photograph of the tumors developed by vector-transfected, HoxB13 or TBX18 expressing cells. C. Tumor weight at the time of sacrifice. D. Western blot analysis of ectopic TBX-18 in the tumors T1 to T4 (shown in A) with anti-Flag antibody.

    Techniques Used: Mouse Assay, Injection, Plasmid Preparation, Transfection, Expressing, Western Blot

    HOXB13 and TBX18 demonstrate anti-tumorignic property in colon cancer cells in vitro. A. Western blot analysis of cell extracts prepared from RKO cells expressing Flag-tagged HOXB13 or TBX18. Analysis of cell growth by MTT assay ( B ), replication potential by 3 H 1 -thymidine incorporation ( C ), clonogenic survival ( D ) of RKO cells (pool and a clone selected at random). Each assay was performed in triplicate. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively.
    Figure Legend Snippet: HOXB13 and TBX18 demonstrate anti-tumorignic property in colon cancer cells in vitro. A. Western blot analysis of cell extracts prepared from RKO cells expressing Flag-tagged HOXB13 or TBX18. Analysis of cell growth by MTT assay ( B ), replication potential by 3 H 1 -thymidine incorporation ( C ), clonogenic survival ( D ) of RKO cells (pool and a clone selected at random). Each assay was performed in triplicate. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively.

    Techniques Used: In Vitro, Western Blot, Expressing, MTT Assay

    5) Product Images from "Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling"

    Article Title: Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling

    Journal: bioRxiv

    doi: 10.1101/2020.01.06.896365

    Vpr inhibits ubiquitination of histone H1 variants by CRL4 DCAF1 (A) Schematic of histone h1 protein domains (top; positions indicated are for histone H1.2). Heatmap of phosphorylation (P) and ubiquitination (U) changes on histone H1 variants. All sites are aligned to the sequence positions of histone H1.2. Cells with a red outline were identified by peptides that cannot distinguish between histone variants. (B) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with Vpr titration (C) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with MLN4924 titration (D) Schematic of experimental and data analysis workflows for quantitative affinity purification and mass spectrometry analysis of histone H1.2 in the presence and absence of Vpr co-expression (E) Volcano plot of histone H1.2 protein binding changes in response to Vpr expression. Proteins with |log 2 fold-change| > 0.58 (i.e., 1.5-fold change) and adjusted p-value
    Figure Legend Snippet: Vpr inhibits ubiquitination of histone H1 variants by CRL4 DCAF1 (A) Schematic of histone h1 protein domains (top; positions indicated are for histone H1.2). Heatmap of phosphorylation (P) and ubiquitination (U) changes on histone H1 variants. All sites are aligned to the sequence positions of histone H1.2. Cells with a red outline were identified by peptides that cannot distinguish between histone variants. (B) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with Vpr titration (C) Denaturing ubiquitin immunoprecipitation analysis of histone H1.2 with MLN4924 titration (D) Schematic of experimental and data analysis workflows for quantitative affinity purification and mass spectrometry analysis of histone H1.2 in the presence and absence of Vpr co-expression (E) Volcano plot of histone H1.2 protein binding changes in response to Vpr expression. Proteins with |log 2 fold-change| > 0.58 (i.e., 1.5-fold change) and adjusted p-value

    Techniques Used: Sequencing, Immunoprecipitation, Titration, Affinity Purification, Mass Spectrometry, Expressing, Protein Binding

    6) Product Images from "MARK3-mediated phosphorylation of ARHGEF2 couples the actin and tubulin cytoskeletons to establish cell polarity"

    Article Title: MARK3-mediated phosphorylation of ARHGEF2 couples the actin and tubulin cytoskeletons to establish cell polarity

    Journal: Science signaling

    doi: 10.1126/scisignal.aan3286

    Interaction networks of ARHGEF2 and MARK3. ), and reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. ). Reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. (C) Pyo-tagged wild-type MARK3 and CNK1 (negative control) were co-expressed along with CLASP1, CLASP2, and ARHGEF2 and immunoprecipitated from Cos cell lysates. The protein complexes were examined by Western blot using specific antibodies for CLASP 1, CLASP2 and ARHGEF2 and Pyo for MARK3. (D) Cell lysates from HEK293T were immunoprecipitated using IgG or an antibody recognizing ARHGEF2 combined with Sepharose beads. The protein complexes were separated by SDS page and probed with antibodies recognizing MARK3 or ARHGEF2. Whole cell lysates were analyzed by Western blots to assess MARK3 and ARHGEF2 protein abundance. (E) KSR1 N’ (N-terminal head domain of KSR1), CLASP1, CLASP2 and ARHGEF2 proteins were immunoprecipitated from Cos cells and incubated with purified active wild type (WT) or kinase-dead (KD) MARK3 in the presence of 32 P-ATP. The labeled proteins were separated by SDS-PAGE and visualized by autoradiography. The membrane was then probed for MARK3 to detect the purified MARK3 proteins. (C-E) Representative of three independent experiments. (F) Analysis of the protein sequences of CLASP1, CLASP2 and ARHGEF2 for the consensus MARK3 phosphorylation motifs Φ a xRxxS*ΦPxxΦ a and Φ a xR/KxxS*xxxΦ a using ScanProsite. (S* is the site phosphorylated, x any amino acid, Φ a is a hydrophobic residue with an aliphatic side chain and Φ is any hydrophobic amino acid).
    Figure Legend Snippet: Interaction networks of ARHGEF2 and MARK3. ), and reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. ). Reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. (C) Pyo-tagged wild-type MARK3 and CNK1 (negative control) were co-expressed along with CLASP1, CLASP2, and ARHGEF2 and immunoprecipitated from Cos cell lysates. The protein complexes were examined by Western blot using specific antibodies for CLASP 1, CLASP2 and ARHGEF2 and Pyo for MARK3. (D) Cell lysates from HEK293T were immunoprecipitated using IgG or an antibody recognizing ARHGEF2 combined with Sepharose beads. The protein complexes were separated by SDS page and probed with antibodies recognizing MARK3 or ARHGEF2. Whole cell lysates were analyzed by Western blots to assess MARK3 and ARHGEF2 protein abundance. (E) KSR1 N’ (N-terminal head domain of KSR1), CLASP1, CLASP2 and ARHGEF2 proteins were immunoprecipitated from Cos cells and incubated with purified active wild type (WT) or kinase-dead (KD) MARK3 in the presence of 32 P-ATP. The labeled proteins were separated by SDS-PAGE and visualized by autoradiography. The membrane was then probed for MARK3 to detect the purified MARK3 proteins. (C-E) Representative of three independent experiments. (F) Analysis of the protein sequences of CLASP1, CLASP2 and ARHGEF2 for the consensus MARK3 phosphorylation motifs Φ a xRxxS*ΦPxxΦ a and Φ a xR/KxxS*xxxΦ a using ScanProsite. (S* is the site phosphorylated, x any amino acid, Φ a is a hydrophobic residue with an aliphatic side chain and Φ is any hydrophobic amino acid).

    Techniques Used: Negative Control, Immunoprecipitation, Western Blot, SDS Page, Incubation, Purification, Labeling, Autoradiography

    Structural characterization of the DYNLT1-ARHGEF2 interaction. (A) Fluorescence polarization binding assays were performed with FITC-labelled ARHGEF2 peptides (142–157) with and without phosphorylation of Ser 151 . The peptides were titrated with increasing amounts of recombinant GST-DYNLT1. Representative of two independent experiments. (B) Overlay of ‘H- 15 N HSQC spectra of DYNLT1 in the absence (blue) or presence (red) of mARHGEF2 peptide (136–164), or in the context of a DYNLT1-mARHGEF2 chimera with a single glycine linker (black). Inset boxes (a, b) zoom into the overlay of the spectra. Chemical shift changes for selected residues are highlighted with arrows in the spectra. . (D) Schematic representation of interactions between the domain-swapped P-strand of DYNLT1 and ARHGEF2. Residues of DYNLT1 (grey) and ARHGEF2 (yellow), with the hydrogen bond network are shown by black dotted lines. A kink in ARHGEF2 is caused by the insertion (relative to a perfect β-strand) of residues Val 150 and Ser 151 (cyan box), which form a β-bulge. (E) Detail of DYNLT1 in complex with ARHGEF2. Enlargement showing the ribbon representation of DYNLT1 (blue) with a sticks model of the mARHGEF2 (yellow) component of the chimera (PDB: 5WI4). ARHGEF2 residues Leu 146 to Asn 154 are highlighted.
    Figure Legend Snippet: Structural characterization of the DYNLT1-ARHGEF2 interaction. (A) Fluorescence polarization binding assays were performed with FITC-labelled ARHGEF2 peptides (142–157) with and without phosphorylation of Ser 151 . The peptides were titrated with increasing amounts of recombinant GST-DYNLT1. Representative of two independent experiments. (B) Overlay of ‘H- 15 N HSQC spectra of DYNLT1 in the absence (blue) or presence (red) of mARHGEF2 peptide (136–164), or in the context of a DYNLT1-mARHGEF2 chimera with a single glycine linker (black). Inset boxes (a, b) zoom into the overlay of the spectra. Chemical shift changes for selected residues are highlighted with arrows in the spectra. . (D) Schematic representation of interactions between the domain-swapped P-strand of DYNLT1 and ARHGEF2. Residues of DYNLT1 (grey) and ARHGEF2 (yellow), with the hydrogen bond network are shown by black dotted lines. A kink in ARHGEF2 is caused by the insertion (relative to a perfect β-strand) of residues Val 150 and Ser 151 (cyan box), which form a β-bulge. (E) Detail of DYNLT1 in complex with ARHGEF2. Enlargement showing the ribbon representation of DYNLT1 (blue) with a sticks model of the mARHGEF2 (yellow) component of the chimera (PDB: 5WI4). ARHGEF2 residues Leu 146 to Asn 154 are highlighted.

    Techniques Used: Fluorescence, Binding Assay, Recombinant

    LKB1-MARK3 axis and PP2A regulate the phosphorylation of ARHGEF2 Ser 151 . (A) Western blot of HEK293T cells treated with DMSO, PP2A inhibitor okadaic acid (OA, 50 nM for 4 hours) and AMPK activator AICAR (1mM for 6 hours). Phosphorylated ARHGEF2 Ser 151 for details). Lower panel: quantification of the phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independedent experiments. (B) Western blot of HEK293T cells overexpressing pyo-tagged MARK3 WT or Flag-tagged PPP2R5B. Phosphorylated ARHGEF2 Ser 151 and Ser 885 were detected using site-specific antibodies and a-tubulin was used as a loading control. Lower panel: quantification of Ser 151 and Ser 885 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments. (C) Western blot of 293 T-Rex Flp-In cell lines carrying inducible expression of Flag-tagged GFP, PP2A catalytic subunit PPP2CB or regulatory B’subunit PPP2R5B. The cells were induced overnight with tetracycline 500ng/ml and also transfected with empty vector, pyo-MARK3 WT or pyo-MARK3 KD . Phosphorylated ARHGEF2 Ser 151 was detected using a site-specific antibody and α-tubulin was used as a loading control. Lower panel: quantification of the phosphorylation normalized with total ARHGEF2, Data are means ± SD of four independent experiments. (D) Western blot of A549 LKB1-deficient cells stably expressing empty vector (pBabe), wild-type LKB1 (LKB1 WT ) or kinase deficient LKB1 (LKB1 KD ). Phosphorylation of AMPK was used as a control substrate for LKB1 phosphorylation. Phosphorylated ARHGEF2 Ser 151 and AMPK Thr 172 were detected using site-specific antibodies and GAPDH was used as a loading control. Lower panel: quantification of ARHGEF2 Ser 151 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments. (E) Western blot of A549 cells expressing LBK1 WT and treated with an siRNA pool specific for MARK3 versus control for 72 hours. Phosphorylation AMPK was used as a control substrate for LKB 1 phosphorylation. GAPDH was used as a loading control. Lower panel: quantification of ARHGEF2 Ser 151 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments.
    Figure Legend Snippet: LKB1-MARK3 axis and PP2A regulate the phosphorylation of ARHGEF2 Ser 151 . (A) Western blot of HEK293T cells treated with DMSO, PP2A inhibitor okadaic acid (OA, 50 nM for 4 hours) and AMPK activator AICAR (1mM for 6 hours). Phosphorylated ARHGEF2 Ser 151 for details). Lower panel: quantification of the phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independedent experiments. (B) Western blot of HEK293T cells overexpressing pyo-tagged MARK3 WT or Flag-tagged PPP2R5B. Phosphorylated ARHGEF2 Ser 151 and Ser 885 were detected using site-specific antibodies and a-tubulin was used as a loading control. Lower panel: quantification of Ser 151 and Ser 885 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments. (C) Western blot of 293 T-Rex Flp-In cell lines carrying inducible expression of Flag-tagged GFP, PP2A catalytic subunit PPP2CB or regulatory B’subunit PPP2R5B. The cells were induced overnight with tetracycline 500ng/ml and also transfected with empty vector, pyo-MARK3 WT or pyo-MARK3 KD . Phosphorylated ARHGEF2 Ser 151 was detected using a site-specific antibody and α-tubulin was used as a loading control. Lower panel: quantification of the phosphorylation normalized with total ARHGEF2, Data are means ± SD of four independent experiments. (D) Western blot of A549 LKB1-deficient cells stably expressing empty vector (pBabe), wild-type LKB1 (LKB1 WT ) or kinase deficient LKB1 (LKB1 KD ). Phosphorylation of AMPK was used as a control substrate for LKB1 phosphorylation. Phosphorylated ARHGEF2 Ser 151 and AMPK Thr 172 were detected using site-specific antibodies and GAPDH was used as a loading control. Lower panel: quantification of ARHGEF2 Ser 151 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments. (E) Western blot of A549 cells expressing LBK1 WT and treated with an siRNA pool specific for MARK3 versus control for 72 hours. Phosphorylation AMPK was used as a control substrate for LKB 1 phosphorylation. GAPDH was used as a loading control. Lower panel: quantification of ARHGEF2 Ser 151 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments.

    Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation, Stable Transfection

    MARK3 perturbs the interaction between DYNLT1 and ARHGEF2 and stimulates exchange activity. (A) Microscale thermophoresis binding assays of phosphorylated and unphosphorylated FITC-labelled ARHGEF2 peptides for Ser 885 (amino acids 876–891, top panel) and Ser 151 (amino acids 142–157, lower panel). The peptides were prepared at 100 nM with increasing concentrations of GST-14-3-3. K d (dissociation constant) values were determined from the thermophoresis titration curves for the phosphorylated peptides, whereas no binding was detected for unphosphorylated peptides. Representative of three independent experiments, K d values are average of three independent experiments ±SD. (B) GFP-tagged wild-type ARHGEF2 and a truncated version (deletion of residues 87–151) were co-expressed with Pyo-tagged wild-type MARK3 in HEK293T cells. Protein complexes were immunoprecipitated using an antibody specific for GFP and immunoblots were probed with antibodies recognizing Pyo to detect interactions with MARK3. Antibodies specific for GFP and Pyo were used to detect protein abundance in whole cell lysates. Lower panel: quantification of the interaction normalized with total lysate. Data are means ± SD of three independent experiments. . . Representative of three independent experiments. (E) Myc-tagged wild-type and phosphomimetic mutants S151D and S151E for ARHGEF2 were co-expressed with Flag-tagged DYNLT1. Protein complexes were immunoprecipitated with an antibody recognizing Myc and analyzed by Western blot. Antibodies against Myc, Flag and 14-3-3-were used to confirm the amount of ARHGEF2 and to detect DYNLT1 and endogenous 14-3-3 in the complexes respectively. Protein abundance in whole cell lysates was analyzed using the same antibodies, and a-tubulin was used as a loading control. Representative of three independent experiments. (F) NMR-based GEF assays were performed to measure RHOA exchange rates in the presence of cell lysates from HEK293T cells expressing GFP alone; GFP-ARHGEF2; GFP-ARHGEF2 and Pyo-MARK3; or GFP-ARHGEF2, Pyo-MARK3 and Flag-14-3-3. The amount of ARHGEF2 in exchange assays was normalized on the basis of GFP fluorescence in the lysate and protein expression concentration was detected by Western blot (inset). The rates were normalized to ARHGEF2 exchange rate. Data are means ± SD of five independent experiments. Statistical significance was determined by a Kruskal-Walis test with a Dunn’s post-test correction for multiple comparisons. *P=0.0151 (ARHGEF2 VS ARHGEF2+MARK3); **P=0.0072 (ARHGEF2 VS ARHGEF2+MARK3+14-3-3); **P=0.0079 (GFP VS ARHGEF2). NS: not significant. (G) Nucleotide exchange rates for RHOA in the presence of cell lysates from HEK293T cells expressing GFP-ARHGEF2 wt or GFP-tagged ARHGEF2 S151A . The rates were normalized to ARHGEF2 WT exchange rate. Data are means ± SD of four independent experiments. Statistical significance was determined by a Mann-Whitney test. *P=0.0286.
    Figure Legend Snippet: MARK3 perturbs the interaction between DYNLT1 and ARHGEF2 and stimulates exchange activity. (A) Microscale thermophoresis binding assays of phosphorylated and unphosphorylated FITC-labelled ARHGEF2 peptides for Ser 885 (amino acids 876–891, top panel) and Ser 151 (amino acids 142–157, lower panel). The peptides were prepared at 100 nM with increasing concentrations of GST-14-3-3. K d (dissociation constant) values were determined from the thermophoresis titration curves for the phosphorylated peptides, whereas no binding was detected for unphosphorylated peptides. Representative of three independent experiments, K d values are average of three independent experiments ±SD. (B) GFP-tagged wild-type ARHGEF2 and a truncated version (deletion of residues 87–151) were co-expressed with Pyo-tagged wild-type MARK3 in HEK293T cells. Protein complexes were immunoprecipitated using an antibody specific for GFP and immunoblots were probed with antibodies recognizing Pyo to detect interactions with MARK3. Antibodies specific for GFP and Pyo were used to detect protein abundance in whole cell lysates. Lower panel: quantification of the interaction normalized with total lysate. Data are means ± SD of three independent experiments. . . Representative of three independent experiments. (E) Myc-tagged wild-type and phosphomimetic mutants S151D and S151E for ARHGEF2 were co-expressed with Flag-tagged DYNLT1. Protein complexes were immunoprecipitated with an antibody recognizing Myc and analyzed by Western blot. Antibodies against Myc, Flag and 14-3-3-were used to confirm the amount of ARHGEF2 and to detect DYNLT1 and endogenous 14-3-3 in the complexes respectively. Protein abundance in whole cell lysates was analyzed using the same antibodies, and a-tubulin was used as a loading control. Representative of three independent experiments. (F) NMR-based GEF assays were performed to measure RHOA exchange rates in the presence of cell lysates from HEK293T cells expressing GFP alone; GFP-ARHGEF2; GFP-ARHGEF2 and Pyo-MARK3; or GFP-ARHGEF2, Pyo-MARK3 and Flag-14-3-3. The amount of ARHGEF2 in exchange assays was normalized on the basis of GFP fluorescence in the lysate and protein expression concentration was detected by Western blot (inset). The rates were normalized to ARHGEF2 exchange rate. Data are means ± SD of five independent experiments. Statistical significance was determined by a Kruskal-Walis test with a Dunn’s post-test correction for multiple comparisons. *P=0.0151 (ARHGEF2 VS ARHGEF2+MARK3); **P=0.0072 (ARHGEF2 VS ARHGEF2+MARK3+14-3-3); **P=0.0079 (GFP VS ARHGEF2). NS: not significant. (G) Nucleotide exchange rates for RHOA in the presence of cell lysates from HEK293T cells expressing GFP-ARHGEF2 wt or GFP-tagged ARHGEF2 S151A . The rates were normalized to ARHGEF2 WT exchange rate. Data are means ± SD of four independent experiments. Statistical significance was determined by a Mann-Whitney test. *P=0.0286.

    Techniques Used: Activity Assay, Microscale Thermophoresis, Binding Assay, Titration, Immunoprecipitation, Western Blot, Nuclear Magnetic Resonance, Expressing, Fluorescence, Concentration Assay, MANN-WHITNEY

    MARK3 binds an N-terminal region of ARHGEF2 and phosphorylates Ser 151 . (A) Left: Flag-tagged ARHGEF2 fragments were co-expressed in HEK293T cells with wild-type MARK3. Protein complexes were immunoprecipitated and immunoblots were probed with antibodies specific for MARK3 and pan 14-3-3 to map the interaction. Antibodies recognizing Flag, MARK and 14-3-3 antibodies were used to detect protein abundance in cell lysates; α-tubulin was used as a loading control. Right: Schematic representation of the constructs used for mapping the interaction. (B, C) ARHGEF2 wild-type (top panels) or mutant ARHGEF2 S151A (lower panels) proteins were incubated with 32 P-ATP, digested with trypsin and examined by HPLC analysis. For the in vitro analysis (B) purified MARK3 was added. (D) Purified ARHGEF2 mutants were incubated with purified active MARK3 in the presence of 32 P-ATP. The phosphorylated mutants were separated by SDS-PAGE. For each mutant the 32 P-phosphate incorporated was quantitated using a phosphoimager. (A-D) Representative of three independent experiments. .
    Figure Legend Snippet: MARK3 binds an N-terminal region of ARHGEF2 and phosphorylates Ser 151 . (A) Left: Flag-tagged ARHGEF2 fragments were co-expressed in HEK293T cells with wild-type MARK3. Protein complexes were immunoprecipitated and immunoblots were probed with antibodies specific for MARK3 and pan 14-3-3 to map the interaction. Antibodies recognizing Flag, MARK and 14-3-3 antibodies were used to detect protein abundance in cell lysates; α-tubulin was used as a loading control. Right: Schematic representation of the constructs used for mapping the interaction. (B, C) ARHGEF2 wild-type (top panels) or mutant ARHGEF2 S151A (lower panels) proteins were incubated with 32 P-ATP, digested with trypsin and examined by HPLC analysis. For the in vitro analysis (B) purified MARK3 was added. (D) Purified ARHGEF2 mutants were incubated with purified active MARK3 in the presence of 32 P-ATP. The phosphorylated mutants were separated by SDS-PAGE. For each mutant the 32 P-phosphate incorporated was quantitated using a phosphoimager. (A-D) Representative of three independent experiments. .

    Techniques Used: Immunoprecipitation, Western Blot, Construct, Mutagenesis, Incubation, High Performance Liquid Chromatography, In Vitro, Purification, SDS Page

    MARK3 phosphorylation of ARHGEF2 Ser 151 regulates several biological functions. (A, B) Immunofluorescence of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT , pLVX-GFP ARHGEF2 S151A and pLVX-GFP ARHGEF2 WT in combination with siMARK3. The cells were fixed and stained for actin (left panel), GFP signal is shown (inset); right panel, LUTs showing the fluorescence intensity, white being the more intense. In B, quantification of the Mean Fluorescence intensity of the images shown in A. Five high magnification fields per condition and per experiment were quantified using ImageJ. Data are means ± SD of three independent experiments. Scale bar 20 μm. Statistical significance was determined by a one way ANOVA test with a Bonferroni post-test correction for multiple comparisons. *P=0.0453; ****P
    Figure Legend Snippet: MARK3 phosphorylation of ARHGEF2 Ser 151 regulates several biological functions. (A, B) Immunofluorescence of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT , pLVX-GFP ARHGEF2 S151A and pLVX-GFP ARHGEF2 WT in combination with siMARK3. The cells were fixed and stained for actin (left panel), GFP signal is shown (inset); right panel, LUTs showing the fluorescence intensity, white being the more intense. In B, quantification of the Mean Fluorescence intensity of the images shown in A. Five high magnification fields per condition and per experiment were quantified using ImageJ. Data are means ± SD of three independent experiments. Scale bar 20 μm. Statistical significance was determined by a one way ANOVA test with a Bonferroni post-test correction for multiple comparisons. *P=0.0453; ****P

    Techniques Used: Immunofluorescence, Stable Transfection, Expressing, Staining, Fluorescence

    Phosphorylation of ARHGEF2 Ser 151 is required for normal cell polarity (A-C) 3D culture of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT and pLVX-GFP ARHGEF2 S151A . In (A), GFP fluorescence was visualized and cysts were stained for E-CADHERIN, ACTIN and DAPI. In (B), average size of the cysts observed in pLVX-GFP, pLVX-GFP ARHGEF2 WT and pLVX-GFP ARHGEF2 S151A (n=24; 21; 24 respectively). Data are are means ± SD of three independent experiments. Statistical significance was determined by a one way ANOVA test with a Bonferroni post-test correction for multiple comparisons. ****P≤0.0001. Scale bar, 20 μm. In (C) 1 μm Z-stacks of pLVX-GFP ARHGEF2 WT -cysts. Abnormal mitotic events are indicated (yellow arrows). Note that the cyst on the left has lost expression of pLVX-GFP ARHGEF2 wt . Numbers represent the Z-stack step. Images are representative of four independent experiments. Scale bar, 20 μm. (D) Schematic representation summarizing the effects of MARK3 and PP2A in the regulation of ARHGEF2 phosphorylation and its effects on RHOA activation. LKB1 activates MARK3 that in turn phosphorylates ARHGEF2 on Ser 151 . This creates a 14-3-3 binding site that disrupts ARHGEF2 interaction with DYNLT1 and releases it from microtubules to activate RHOA and formation of stress fibers and focal adhesions. MARK3 phosphorylation of Ser 151 is required for epithelial cell polarit y in three-dimensional growth. PP2A dephosphorylates Ser 151 through interactions with the B’ subunits.
    Figure Legend Snippet: Phosphorylation of ARHGEF2 Ser 151 is required for normal cell polarity (A-C) 3D culture of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT and pLVX-GFP ARHGEF2 S151A . In (A), GFP fluorescence was visualized and cysts were stained for E-CADHERIN, ACTIN and DAPI. In (B), average size of the cysts observed in pLVX-GFP, pLVX-GFP ARHGEF2 WT and pLVX-GFP ARHGEF2 S151A (n=24; 21; 24 respectively). Data are are means ± SD of three independent experiments. Statistical significance was determined by a one way ANOVA test with a Bonferroni post-test correction for multiple comparisons. ****P≤0.0001. Scale bar, 20 μm. In (C) 1 μm Z-stacks of pLVX-GFP ARHGEF2 WT -cysts. Abnormal mitotic events are indicated (yellow arrows). Note that the cyst on the left has lost expression of pLVX-GFP ARHGEF2 wt . Numbers represent the Z-stack step. Images are representative of four independent experiments. Scale bar, 20 μm. (D) Schematic representation summarizing the effects of MARK3 and PP2A in the regulation of ARHGEF2 phosphorylation and its effects on RHOA activation. LKB1 activates MARK3 that in turn phosphorylates ARHGEF2 on Ser 151 . This creates a 14-3-3 binding site that disrupts ARHGEF2 interaction with DYNLT1 and releases it from microtubules to activate RHOA and formation of stress fibers and focal adhesions. MARK3 phosphorylation of Ser 151 is required for epithelial cell polarit y in three-dimensional growth. PP2A dephosphorylates Ser 151 through interactions with the B’ subunits.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Staining, Activation Assay, Binding Assay

    MARK3 affects the localization of ARHGEF2 dependent on Ser 151 and 14-3-3. (A, B) Live imaging pictures of HEK293T cells. In A, top panel, cells transiently overexpressing GFP alone, GFP-ARHGEF2 wt alone or in combination with Cherry-MARK3; bottom panel cells expressing GFP- ARHGEF2 S151A alone or co-expressed with Cherry-MARK3. ARHGEF2 distribution in A is quantified in B (as percentage of cells). A total of n=100–200 cells per condition were counted. Data are means ± SD of three independent experiments (n=3). Scale bar, 10 μm. Statistical significance was determined by a two way ANOVA test with a Bonferroni posttest correction for multiple comparisons. ****P≤0.0001. (C, D) Live imaging of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT , pLVX-GFP ARHGEF2 S151A and pLVX-GFP ARHGEF2 WT in combination with siMARK3; zoomed regions shown in bottom panels. In (D) quantification, as percentage of cells, of images shown in (C): cells having a higher tendency of showing a filament-like distribution (F > D); a higher tendency of having a diffuse distribution (D > F) or similar distribution of filament-like and diffuse appearing structures (D=F). F (Filament-Like distribution); D (Diffuse distribution). A total of n=250–300 cells per condition were counted. Data are means ± SD of three independent experiments. Scale bar 20 μm (upper images), 10 μm (lower images). Statistical significance was determined by a two way ANOVA test with a Bonferroni post-test correction for multiple comparisons. *P=0.0470 (in F > D ARHGEF2 wt VS ARHGEF2 WT +siMARK3); *P=0.0235 (in F=D ARHGEF2 WT VS ARHGEF2 WT +siMARK3); **P=0.0014; ***P=0.0002 (in F > D ARHGEF2 WT + siMARK3 VS ARHGEF2 S151A ); ***P=0.0005 (in D > F ARHGEF2 wt VS ARHGEF2 WT +siMARK3); ****P≤0.0001.
    Figure Legend Snippet: MARK3 affects the localization of ARHGEF2 dependent on Ser 151 and 14-3-3. (A, B) Live imaging pictures of HEK293T cells. In A, top panel, cells transiently overexpressing GFP alone, GFP-ARHGEF2 wt alone or in combination with Cherry-MARK3; bottom panel cells expressing GFP- ARHGEF2 S151A alone or co-expressed with Cherry-MARK3. ARHGEF2 distribution in A is quantified in B (as percentage of cells). A total of n=100–200 cells per condition were counted. Data are means ± SD of three independent experiments (n=3). Scale bar, 10 μm. Statistical significance was determined by a two way ANOVA test with a Bonferroni posttest correction for multiple comparisons. ****P≤0.0001. (C, D) Live imaging of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2 WT , pLVX-GFP ARHGEF2 S151A and pLVX-GFP ARHGEF2 WT in combination with siMARK3; zoomed regions shown in bottom panels. In (D) quantification, as percentage of cells, of images shown in (C): cells having a higher tendency of showing a filament-like distribution (F > D); a higher tendency of having a diffuse distribution (D > F) or similar distribution of filament-like and diffuse appearing structures (D=F). F (Filament-Like distribution); D (Diffuse distribution). A total of n=250–300 cells per condition were counted. Data are means ± SD of three independent experiments. Scale bar 20 μm (upper images), 10 μm (lower images). Statistical significance was determined by a two way ANOVA test with a Bonferroni post-test correction for multiple comparisons. *P=0.0470 (in F > D ARHGEF2 wt VS ARHGEF2 WT +siMARK3); *P=0.0235 (in F=D ARHGEF2 WT VS ARHGEF2 WT +siMARK3); **P=0.0014; ***P=0.0002 (in F > D ARHGEF2 WT + siMARK3 VS ARHGEF2 S151A ); ***P=0.0005 (in D > F ARHGEF2 wt VS ARHGEF2 WT +siMARK3); ****P≤0.0001.

    Techniques Used: Imaging, Expressing, Stable Transfection

    Related Articles

    Transfection:

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    Protease Inhibitor:

    Article Title: AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors
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    Flow Cytometry:

    Article Title: Barttin modulates trafficking and function of ClC-K channels
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    Cell Culture:

    Article Title: Dynamic L-type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating
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    Cytometry:

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    Incubation:

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    other:

    Article Title: AMPAR/TARP stoichiometry differentially modulates channel properties
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    Recombinant:

    Article Title: Mechanisms of CPT1C-Dependent AMPAR Trafficking Enhancement
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    Plasmid Preparation:

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