anti mypt1 rabbit polyclonal  (Santa Cruz Biotechnology)

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Interaction of MYPT1 with Rho-kinase. ( A ) Schematic representation of the domain structure and deletion constructs of MYPT1. ( B , C ) Mapping of the Rho-kinase-interacting region of myosin phosphatase targeting subunit 1 (MYPT1). COS-7 cells were cotransfected with the GST-MYPT1 fragment and GFP-Rho-kinase-cat-L (6–553 aa) and pulled-down with glutathione beads. The bound proteins were subjected to immunoblot analysis using an anti-GST or anti-GFP antibody. The C-terminus of MYPT1 bound to Rho-kinase-cat-L, in which phosphorylation sites of MYPT1 contributed to the binding. These results are representative of at least three independent experiments.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Construct, Western Blot, Binding Assay

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Identification of the docking motifs of MYPT1. ( A , B ) Identification of the regions/sites responsible for the binding to Rho-kinase. COS-7 cells were cotransfected with the GST-MYPT1 fragment with deletion or amino acid substitution and GFP-Rho-kinase-cat (6–418 aa) and pulled-down with glutathione beads. The bound proteins were subjected to immunoblot analysis using an anti-GST or anti-GFP antibody. The deletions of docking motif (DM)1 (704–715 aa) and DM2 (836–849 aa) and/or substitutions of phosphorylation sites diminished the association with Rho-kinase. These results are representative of at least three independent experiments.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Binding Assay, Western Blot

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Inhibition of MYPT1 phosphorylation by the Rho-kinase-binding fragment of MYPT1. ( A , B ) Effect of the overexpression of Rho-kinase-binding MYPT1 fragments on GFP-MYPT1 phosphorylation in COS7 cells. Full-length GFP-MYPT1 and the GST-MYPT1 Rho-kinase binding fragment were transiently expressed in COS7 cells. The cell lysates were analyzed by immunoblot analysis using anti-MYPT1-pT855 (top panel), anti-GFP (middle), or anti-GST (bottom) antibodies. The phospho-GFP-MYPT1 levels were quantified and normalized by GFP-MYPT1 levels. Data represent means ± SD. **, p < 0.01, ***, p < 0.001 as compared with the control. ( C ) Effect of the overexpression of Rho-kinase-binding MYPT1 fragments on stress fiber formation in NIH3T3 cells. GST-MYPT1 fragments were expressed in NIH3T3 cells. The cells were fixed in formaldehyde, and immunostained with phalloidin and anti-GST antibody. Colors indicate GST (green) and F-actin (red). Scale bar, 10 μm. The percentages of cells with stress fibers throughout the cell (SF(++)), cells with stress fibers partly in the cell (SF(+)), and cells without stress fibers (SF(–)) were analyzed. Data are means ± SD. N = 4. ***, p < 0.001 as compared with control. DN-RhoK: dominant negative Rho-kinase.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Inhibition, Binding Assay, Over Expression, Western Blot, Dominant Negative Mutation

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Direct inhibition of Rho-kinase activity in vitro. ( A ) In vitro phosphorylation of S6 rsk substrate peptide by Rho-kinase. GST-Rho-kinase-cat-L or GST-ROCK1-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. GST-MYPT1-684–868 aa-WT or -4A was added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. ( B ) Effects of the deletion of DMs on the inhibition of Rho-kinase activity in vitro. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. GST-MYPT1-684–868-4A with or without DMs was added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. The deletion of DM1 or DM2 moderately weakened the inhibitory effect and the deletion of both DM1 and DM2 completely abrogated the inhibitory effects of the MYPT1 fragment on the Rho-kinase activity. ( C ) Sequences of the DMs and phosphorylation sites of MYPT1. The sequences of MYPT1 683–715 aa and 836-860 aa and synthetic peptides are shown. Basic (blue), acidic (red), and aliphatic (green) amino acids within DMs are indicated. Phosphorylation sites are replaced by Ala in pseudosubstrate (PS) peptides. ( D ) Inhibition of Rho-kinase activity by PS2 + DM2 synthetic peptide. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. Synthetic peptides were added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. ( E ) Inhibition of Rho-kinase activity by PS1 + DM3 synthetic peptide. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. Synthetic peptides were added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Inhibition, Activity Assay, In Vitro

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Effects of the DMs on MYPT1 phosphorylation. ( A ) Suppression of MYPT1 fragment phosphorylation by deletion of DMs in vitro. One μM of GST-MYPT1-fragments was phosphorylated by 1 nM of Rho-kinase-cat-L in the presence of γ-[ 32 P]ATP. Phosphorylation levels of GST-MYPT1-684–758 aa-ΔDM1, -690–758 aa (deletion of DM3 from 684–758 aa fragment), and -690–758 aa-ΔDM1 (deletion of both DM1 and DM3 from 684–758 aa fragment) were compared with that of GST-MYPT1-684–758 aa, and the phosphorylation level of GST-MYPT1-758-868 aa-ΔDM2 was compared with that of GST-MYPT1-758–868 aa. Data represent means ± SD. N = 3. ***, p < 0.001. ( B ) Suppression of MYPT1 phosphorylation by deletion of DMs in COS7 cells. GFP-MYPT1-WT or mutants were transiently expressed with or without GST-Rho-kinase-cat-L in COS7 cells. The cell lysates were analyzed by immunoblot analysis using anti-MYPT1-pT855, anti-MYPT1-pS854, anti-MYPT1-pT697, or anti-GFP antibodies. The phospho-GFP-MYPT1 levels were quantified and normalized by GFP-MYPT1 levels. Data represent means ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001 as compared with the control.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: In Vitro, Western Blot

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: ( A ) Schematic representation of phosphorylation sites and DMs of MYPT1. ( B ) Model for kinase–substrate recognition between Rho-kinase and MYPT1.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques:

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Interaction of MYPT1 with Rho-kinase. ( A ) Schematic representation of the domain structure and deletion constructs of MYPT1. ( B , C ) Mapping of the Rho-kinase-interacting region of myosin phosphatase targeting subunit 1 (MYPT1). COS-7 cells were cotransfected with the GST-MYPT1 fragment and GFP-Rho-kinase-cat-L (6–553 aa) and pulled-down with glutathione beads. The bound proteins were subjected to immunoblot analysis using an anti-GST or anti-GFP antibody. The C-terminus of MYPT1 bound to Rho-kinase-cat-L, in which phosphorylation sites of MYPT1 contributed to the binding. These results are representative of at least three independent experiments.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Construct, Western Blot, Binding Assay

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Identification of the docking motifs of MYPT1. ( A , B ) Identification of the regions/sites responsible for the binding to Rho-kinase. COS-7 cells were cotransfected with the GST-MYPT1 fragment with deletion or amino acid substitution and GFP-Rho-kinase-cat (6–418 aa) and pulled-down with glutathione beads. The bound proteins were subjected to immunoblot analysis using an anti-GST or anti-GFP antibody. The deletions of docking motif (DM)1 (704–715 aa) and DM2 (836–849 aa) and/or substitutions of phosphorylation sites diminished the association with Rho-kinase. These results are representative of at least three independent experiments.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Binding Assay, Western Blot

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Inhibition of MYPT1 phosphorylation by the Rho-kinase-binding fragment of MYPT1. ( A , B ) Effect of the overexpression of Rho-kinase-binding MYPT1 fragments on GFP-MYPT1 phosphorylation in COS7 cells. Full-length GFP-MYPT1 and the GST-MYPT1 Rho-kinase binding fragment were transiently expressed in COS7 cells. The cell lysates were analyzed by immunoblot analysis using anti-MYPT1-pT855 (top panel), anti-GFP (middle), or anti-GST (bottom) antibodies. The phospho-GFP-MYPT1 levels were quantified and normalized by GFP-MYPT1 levels. Data represent means ± SD. **, p < 0.01, ***, p < 0.001 as compared with the control. ( C ) Effect of the overexpression of Rho-kinase-binding MYPT1 fragments on stress fiber formation in NIH3T3 cells. GST-MYPT1 fragments were expressed in NIH3T3 cells. The cells were fixed in formaldehyde, and immunostained with phalloidin and anti-GST antibody. Colors indicate GST (green) and F-actin (red). Scale bar, 10 μm. The percentages of cells with stress fibers throughout the cell (SF(++)), cells with stress fibers partly in the cell (SF(+)), and cells without stress fibers (SF(–)) were analyzed. Data are means ± SD. N = 4. ***, p < 0.001 as compared with control. DN-RhoK: dominant negative Rho-kinase.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Inhibition, Binding Assay, Over Expression, Western Blot, Dominant Negative Mutation

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Direct inhibition of Rho-kinase activity in vitro. ( A ) In vitro phosphorylation of S6 rsk substrate peptide by Rho-kinase. GST-Rho-kinase-cat-L or GST-ROCK1-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. GST-MYPT1-684–868 aa-WT or -4A was added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. ( B ) Effects of the deletion of DMs on the inhibition of Rho-kinase activity in vitro. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. GST-MYPT1-684–868-4A with or without DMs was added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. The deletion of DM1 or DM2 moderately weakened the inhibitory effect and the deletion of both DM1 and DM2 completely abrogated the inhibitory effects of the MYPT1 fragment on the Rho-kinase activity. ( C ) Sequences of the DMs and phosphorylation sites of MYPT1. The sequences of MYPT1 683–715 aa and 836-860 aa and synthetic peptides are shown. Basic (blue), acidic (red), and aliphatic (green) amino acids within DMs are indicated. Phosphorylation sites are replaced by Ala in pseudosubstrate (PS) peptides. ( D ) Inhibition of Rho-kinase activity by PS2 + DM2 synthetic peptide. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. Synthetic peptides were added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3. ( E ) Inhibition of Rho-kinase activity by PS1 + DM3 synthetic peptide. GST-Rho-kinase-cat-L activity was examined using the S6 rsk substrate peptide as a substrate in the presence of γ-[ 32 P]ATP. Synthetic peptides were added at the indicated concentrations. The reaction mixtures were applied onto cation-exchange membranes, followed by scintillation counting. N = 3.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: Inhibition, Activity Assay, In Vitro

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: Effects of the DMs on MYPT1 phosphorylation. ( A ) Suppression of MYPT1 fragment phosphorylation by deletion of DMs in vitro. One μM of GST-MYPT1-fragments was phosphorylated by 1 nM of Rho-kinase-cat-L in the presence of γ-[ 32 P]ATP. Phosphorylation levels of GST-MYPT1-684–758 aa-ΔDM1, -690–758 aa (deletion of DM3 from 684–758 aa fragment), and -690–758 aa-ΔDM1 (deletion of both DM1 and DM3 from 684–758 aa fragment) were compared with that of GST-MYPT1-684–758 aa, and the phosphorylation level of GST-MYPT1-758-868 aa-ΔDM2 was compared with that of GST-MYPT1-758–868 aa. Data represent means ± SD. N = 3. ***, p < 0.001. ( B ) Suppression of MYPT1 phosphorylation by deletion of DMs in COS7 cells. GFP-MYPT1-WT or mutants were transiently expressed with or without GST-Rho-kinase-cat-L in COS7 cells. The cell lysates were analyzed by immunoblot analysis using anti-MYPT1-pT855, anti-MYPT1-pS854, anti-MYPT1-pT697, or anti-GFP antibodies. The phospho-GFP-MYPT1 levels were quantified and normalized by GFP-MYPT1 levels. Data represent means ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001 as compared with the control.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: In Vitro, Western Blot

Journal: Biomolecules

Article Title: Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase

doi: 10.3390/biom12020159

Figure Lengend Snippet: ( A ) Schematic representation of phosphorylation sites and DMs of MYPT1. ( B ) Model for kinase–substrate recognition between Rho-kinase and MYPT1.

Article Snippet: Anti-GFP monoclonal mouse antibody (Roche Diagnostics, Basel, Switzerland), anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody (EMD Millipore, Billerica, MA, USA), and Alexa555-Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) were purchased.

Techniques: