Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (A) ARHGEF2 high-confidence interactors detected by BioID mass spectroscopy (see Table S1). Proteins with roles in similar biological processes are grouped by the indicated functions (see Table S3), and reported protein-protein interactions (GeneMANIA) are highlighted with blue edges.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Mass Spectrometry

Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (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.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Immunoprecipitation, Western Blot, Construct

Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (A) Microscale thermophoresis binding assays of phosphorylated and unphosphorylated FITC-labelled ARHGEF2 peptides for Ser885 (amino acids 876–891, top panel) and Ser151 (amino acids 142–157, lower panel). The peptides were prepared at 100 nM with increasing concentrations of GST-14-3-3. Kd (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, Kd values are average of three independent experiments ±SD.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Microscale Thermophoresis, Binding Assay, Titration

Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (A) Fluorescence polarization binding assays were performed with FITC-labelled ARHGEF2 peptides (142–157) with and without phosphorylation of Ser151. The peptides were titrated with increasing amounts of recombinant GST-DYNLT1. Representative of two independent experiments.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Fluorescence, Binding Assay, Recombinant

Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (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 Ser151 was detected using a site-specific antibody and total ERK was used as a loading control (See Figure S5 for details). Lower panel: quantification of the phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independedent experiments.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Western Blot

Journal: Science signaling

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

doi: 10.1126/scisignal.aan3286

Figure Lengend Snippet: (A, B) Live imaging pictures of HEK293T cells. In A, top panel, cells transiently overexpressing GFP alone, GFP-ARHGEF2wt alone or in combination with Cherry-MARK3; bottom panel cells expressing GFP- ARHGEF2S151A 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.

Article Snippet: Antibodies and reagents Western blotting, immunoprecipitation and immunofluorescence were performed using the following antibodies: an antibody recognizing the Pyo derived epitope tag has been reported previously ( 110 ); antibodies recognizing MARK3 and VINCULIN (immunofluorescence) from Milipore; antibodies against Flag (clone M2, Sigma) and HA (Covance); for detection of endogenous ARHGEF2 we used a mouse monoclonal antibody, clone 3C5, designed using N-terminal human ARHGEF2 peptides and produced by hybridoma as previously described ( 26 ) and an antibody recognizing a region within amino acids 656–1000 of Human ARHGEF2 (Abcam, ab155785); for endogenous immunoprecipitation of ARHGEF2 we used the ARHGEF2 specific antibody A301–929A (Bethyl); antibodies recognizing 14-3-3 (all isoforms), Myc (9E10), alpha tubulin and ZO-2 (immunofluorescence) from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies recognizing ERK1/2, pARHGEF2 Ser 886 , pARHGEF2 Ser 151 (custom produced), AMPKα, pAMPKα Thr 172 , LKB1, GAPDH and Cleaved Caspase 3 (immunofluorescence) from Cell Signaling; Alexa Fluor® 647 Phalloidin from Thermo Fisher Scientific; antibody specific for ZO-1 from Life Technologies; antibody specific for Ki67 from Abcam (immunofluorescence).

Techniques: Imaging, Expressing

Journal: bioRxiv

Article Title: The LUBAC participates in Lysophosphatidic Acid-induced NF-κB Activation

doi: 10.1101/2020.02.13.948125

Figure Lengend Snippet: A, HEK293T were transfected with a vector encoding for GEF-H1 or with an empty vector (EV) together with an NF-κB reporter plasmid. Shown is the mean ± SEM of triplicate experiments. ****P<0.0001 (ANOVA). B, HEK293T cells were transfected with an empty vector (EV), or with a GEF-H1 plasmid together with FLAG-tagged HOIL1-WT or HOIL1 - R165G. Cell lysates were subjected to Western blotting analysis with antibodies specific to the indicated proteins. White and red arrowheads show full-length and cleaved HOIL1, respectively. Molecular weight markers (M r ) are shown. C, MEFs knockout for GEF-H1 ( GEF-H1 -/- ) or control MEFs ( GEF-H1 +/+ ) were stimulated with 10 μg.mL −1 LPA or with 50 ng.mL −1 PMA plus 100 ng.mL −1 Ionomycin (PI) as indicated. Lysates were prepared and Western blotting analysis was performed as indicated. D, Abundance of IL-6, measured by ELISA in the supernatant of cells as in (C) stimulated for 3 hours (means ± SEM, n=3, ****P<0.0001, ANOVA). Data are representative of three independent experiments.

Article Snippet: Antibodies to phosphorylated IκBα (5A5, 1:5,000), and GEF-H1 (55B6, 1:1,000) were from Cell Signaling Technology.

Techniques: Transfection, Plasmid Preparation, Western Blot, Molecular Weight, Knock-Out, Control, Enzyme-linked Immunosorbent Assay