Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Galectin 3 Regulates HCC cell invasion by RhoA and MLCK activation

doi: 10.1038/labinvest.2015.77

Figure Lengend Snippet: Hepa1-6 cells were incubated with Y-27632 (ROCK inhibitor, 10μM), and a wound closure assay was performed. ROCK inhibition resulted in a significant decrease in the migratory rate compared to the control cells. ( A, B ; N=4, *p<0.05). The phosphorylation of MLC2 was examined as downstream target of galectin 3 and RhoA/ROCK by Western blots ( C ). Densitometry based on 3 independent experiments showed that in the galectin 3 siRNA-transfected cells, the phosphorylation of MLC2 was significantly reduced ( D, N=3, *p<0.05).

Article Snippet: The blots were incubated with the appropriate antibodies for 16h at 4 C: anti-phospo-NFκB (p65) (Cell Signaling Techology), phospho MLC2 and MLC2 antibodies (Cell Signaling Technology), phospho-Akt and total Akt antibodies (Cell Signaling Technology) or the anti-galectin 3 antibody.

Techniques: Incubation, Wound Closure Assay, Inhibition, Control, Phospho-proteomics, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ

doi: 10.1074/jbc.RA118.006075

Figure Lengend Snippet: TIMAP interacts directly with the myosin MLC2 subunit. A , colocalization of overexpressed GFP-TIMAP WT ( top panels , green ) and GFP-TIMAP S333A/S337A ( bottom panels , green ) with MLC2 ( red ) in COS7 cells. Scale bars = 10 μm. Areas in white boxes were digitally magnified. B , immunoreactivity of anti-TIMAP ( red ), anti-MLC2 ( top panels , green ), and anti-pThr 18 /Ser 19 MLC2 ( pMLC2 ; bottom panels , green ) antibodies with endogenous proteins in cultured glomerular ECs. Scale bars = 10 μm. Areas in white boxes were digitally magnified. C , endogenous TIMAP immunoprecipitated from glomerular EC lysates ( TLC ) with chicken anti-TIMAP IgY ( IP: α -TIMAP IGY ) or nonimmune IgY ( IP: IGY ), followed by WB analysis for TIMAP, MLC2, and Myosin IIA heavy chain. D , recombinant purified GST or GST-TIMAP immobilized on GSH beads was incubated with recombinant purified His-MLC2 in vitro , followed by precipitation of the beads. Top , Amido Black–stained blot. Bottom , anti-MLC2 WB analysis. E , recombinant His-MLC2 incubated in vitro in kinase buffer with or without MLCK, followed by pulldown with GST-TIMAP and WB analysis for pMLC2 and total MLC2.

Article Snippet: Polyclonal rabbit anti-pThr 18 /pSer 19 MLC2 (catalog no. 3674), monoclonal rabbit anti-MLC2 antibody (catalog no. 8505), and monoclonal rabbit anti-MYPT1 (catalog no. 8574) were from Cell Signaling Technology (New England Biolabs, Inc.).

Techniques: Cell Culture, Immunoprecipitation, Recombinant, Purification, Incubation, In Vitro, Staining

Journal: The Journal of Biological Chemistry

Article Title: TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ

doi: 10.1074/jbc.RA118.006075

Figure Lengend Snippet: TIMAP overexpression increases MLC2 phosphorylation and stress fiber formation in ECs. A , GFP-TIMAP was overexpressed using an adenoviral ( Ad ) vector in glomerular ECs. Left panel , representative WB analysis. Right panel , quantitative analysis (mean ± S.D.; n = 3 independent experiments; *, p < 0.01). B , glomerular ECs were transduced with adenoviral GFP-TIMAP WT or GFP-TIMAP V64A/F66A at 10, 20, and 40 m.o.i., followed 48 h later by WB analysis for TIMAP, pMLC2, pERM, and tubulin. C , quantification of WB analysis from three independent experiments ( black columns , GFP-vector; white columns , GFP-TIMAP WT ; gray columns , GFP-TIMAP V64A/F66A ; *, p < 0.001, two-way ANOVA with post hoc Bonferroni test for individual differences). D , glomerular ECs were transduced with adenoviral GFP-TIMAP WT or GFP-TIMAP V64A/F66A (m.o.i. 20). 48 h later, cells from each group were mixed 50:50 with glomerular ECs that had been transduced with GFP-vector. GFP-TIMAP WT and GFP-TIMAP V64A/F66A expression was observed throughout the cells, whereas GFP alone was nuclear in GFP-vector–transduced cells. pMLC2 immunofluorescence was markedly augmented in ECs expressing GFP-TIMAP WT but not in ECs expressing GFP-TIMAP V64A/F66A . E , glomerular EC were transduced as in D , followed by evaluation for GFP fluorescence and phalloidin-labeled polymerized actin ( red ). Compared with vector-transduced cells, phalloidin labeling of stress fibers was enhanced in ECs expressing GFP-TIMAP WT but not in ECs expressing GFP-TIMAP V64A/F66A . Scale Bars = 10 μm.

Article Snippet: Polyclonal rabbit anti-pThr 18 /pSer 19 MLC2 (catalog no. 3674), monoclonal rabbit anti-MLC2 antibody (catalog no. 8505), and monoclonal rabbit anti-MYPT1 (catalog no. 8574) were from Cell Signaling Technology (New England Biolabs, Inc.).

Techniques: Over Expression, Phospho-proteomics, Plasmid Preparation, Transduction, Expressing, Immunofluorescence, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ

doi: 10.1074/jbc.RA118.006075

Figure Lengend Snippet: TIMAP raises steady-state pMLC2 levels by reducing the rate of pMLC2 dephosphorylation. A , cultured HUVECs were transfected with control or TIMAP-specific siRNA. Left panel , representative WB analysis. Right panel , densitometric analysis ( n = 5 independent experiments; *, p < 0.01). Β, whole-lung lysates from WT ( TIMAP +/+ ) and age-matched TIMAP-deficient ( TIMAP −/− ) mice. Left panel , WB analysis for TIMAP, pMLC2, and MLC2. Each lane represents lysate from a distinct mouse. Right panel , densitometric quantification of pMLC2/MLC2 ( n = 4/group; *, p < 0.05). C , glomerular ECs transduced with GFP-vector or GFP-TIMAP WT treated with thrombin (1.0 units/ml). Left panel , representative WB analysis. Right panel , quantification of densitometric data from four independent experiments: GFP-vector (■) or GFP-TIMAP WT (♦) (mean ± S.D.; *, p < 0.01; two-way ANOVA with post hoc Bonferroni test for differences at distinct time points). D , glomerular ECs transduced with GFP-vector, GFP-TIMAP WT , or GFP-TIMAP V64A/F66A . pMLC2 abundance was assessed as a function of time after addition of the MLCK inhibitor ML7 (50 μ m ). Left panel , representative WB analysis. Right panel , densitometric analysis. Vector, ■; GFP-TIMAP WT , ♦; GFP-TIMAP V64A/F66A , ●; n = 4 independent experiments; *, p < 0.01, vector versus TIMAP WT ; two-way ANOVA with post hoc Bonferroni analysis.

Article Snippet: Polyclonal rabbit anti-pThr 18 /pSer 19 MLC2 (catalog no. 3674), monoclonal rabbit anti-MLC2 antibody (catalog no. 8505), and monoclonal rabbit anti-MYPT1 (catalog no. 8574) were from Cell Signaling Technology (New England Biolabs, Inc.).

Techniques: De-Phosphorylation Assay, Cell Culture, Transfection, Control, Transduction, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: Effects of NaB on the interaction between MLCK and calmodulin as well as phosphorylation levels of MLC2 in Caco-2 cell monolayers. ( A ) After Ca 2+ switch, Caco-2 cells were cultured in normal Caco-2 medium with or without 2 mmol/L of NaB. Co-Immunoprecipitation (Co-IP) of MLCK and calmodulin was performed at 0, 4 or 8 h, respectively; ( B ) The quantification of MLCK immunoreactive signals by normalized to calmodulin signals in ( A ); ( C ) The change of TERs after Ca 2+ switch under the condition of 2 mmol/L of NaB, or 250 μmol/L of Permeant inhibitor of MLC kinase (PIK) at 0, 2, 4 and 8 h, respectively; ( D ) Total cell lysates from untreated cells or those treated with 2 mmol/L of NaB or 250 μmol/L of PIK were subjected to immunoblotting for pSer19-MLC2, total MLC2 and GAPDH, respectively; ( E ) MLC2 activity was expressed as the ratio of the phosphorylated form of the MLC2 to total MLC2. Values are means ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and controls as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference ( p < 0.05) between NaB and NaB/PIK. PIK-Permeant inhibitor of MLC kinase.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Phospho-proteomics, Cell Culture, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: The phosphorylation change of MLC2 and PKCβ2 during TJs assembly induced by NaB. ( A ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 umol/L of SKF-96365; ( B , C ) The ratio of phosphorylated MLC2 to MLC2 or phosphorylated PKCβ2 to PKCβ2 in ( A ); ( D ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 5 μmol/L of LY-333531; ( E ) The ratio of phosphorylated MLC2 to MLC2 in ( D ); ( F ) Western blotting was performed in the condition of medium alone, 2 mmol/L of NaB and 10 μmol/L of Compound C; ( G ) The ratio of phosphorylated MLC2 to MLC2 in ( F ). All experiments were performed at 8 h after calcium switch. Data represent mean ± SE, n = 3. The asterisks denote a significant difference between chemical-treated groups and control group as p < 0.05 by two-factor ANOVA. The # symbol denotes a significant difference between indicated groups, p < 0.05. CC—Compound C.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

doi: 10.3390/ijms17101696

Figure Lengend Snippet: Proposed diagram showing the mechanism of NaB on reassembly of tight junctions in Caco-2 monolayers. NaB appears to activate Store-Operated Ca 2+ Channel (SOCC) which conducts the Ca 2+ influx and then activates CaMKKβ/AMPK, resulting in PKCβ2 and MLCK/MLC2 pathways to mediate barrier function recovery. The black arrows indicate active effects, and the line with bar at the end indicates inhibition effect. The red arrow indicates reduction, and the green arrows indicates increase.

Article Snippet: The target proteins on membranes were respectively blotted using primary antibodies of S19 phospho-MLC2 (1:2000), MLC2 (1:1000), (Cell Signaling Technology, Danvers, MA, USA), PKCβ1 (1:500), PKCβ2 (phosphor S660) (1:50,000, Abcam, USA), p-PKCβ1 (Thr641) (1:200), PKCβ2 (1:200, Santa Cruz, CA, USA), and GAPDH (1:2000, Proteintech, Rosemont, IL, USA).

Techniques: Inhibition

Journal: Nature communications

Article Title: MURC deficiency in smooth muscle attenuates pulmonary hypertension.

doi: 10.1038/ncomms12417

Figure Lengend Snippet: Figure 3 | Attenuation of MYPT1 and MLC2 phosphorylation in the Murc / lung. (a) MYPT1 phosphorylation in WT, Murc / and Cav1 / mice at the age of 16 weeks under normoxic conditions (n ¼ 3 per group). *Po0.05 compared with WT mice, wPo0.05 compared with Murc / mice. (b) The phosphorylation of MLC2 was assessed in the lungs of WT, Murc / , and Cav1 / mice at the age of 16 weeks under normoxic conditions. Lung sections were immunostained with pMLC2Ser19 and aSMA. Scale bar, 50 mm. (c) MYPT1 phosphorylation in WTand Murc / mice under hypoxic conditions (n ¼ 3 per group). **Po0.01 compared with normoxic WTgroup, wPo0.05 compared with hypoxic WTgroup. (d) The phosphorylation of MLC2 was assessed in the lung exposed to hypoxia for 4 weeks. Lung sections were immunostained with pMLC2Ser19 and aSMA. Scale bar, 20 mm. (e) MYPT1 phosphorylation in the lungs of WT and Murc / mice treated with either PBS or Y-27632 (30 mg kg 1 day 1 for 4 weeks) under hypoxic conditions (n ¼ 4–6 per group). *Po0.05 compared with the PBS-treated WT group exposed to hypoxia. (f) MLC2 phosphorylation in the lungs of WT and Murc / mice treated with either PBS or Y-27632 (30 mg kg 1 day 1 for 4 weeks) under hypoxic conditions (n ¼ 3 per group). *Po0.05 compared with the PBS-treated WT group exposed to hypoxia. Data are presented as mean±s.e.m. Uncropped images of blots are shown in Supplementary Fig. 6.

Article Snippet: 10 NATURE COMMUNICATIONS | 7:12417 | DOI: 10.1038/ncomms12417 | www.nature.com/naturecommunications the rat monoclonal antibody to hemagglutinin (HA, 1:500) was from Roche Applied Science; the mouse monoclonal antibody to T7 (1:5,000) was from Novus Biologicals; the horseradish peroxidase-conjugated monoclonal antibody to GST (1:5,000) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); the mouse monoclonal antibody to GAPDH (1:2,000) was from Millipore; the mouse monoclonal antibody to FLAG (1:1,000), Cy3-conjugated monoclonal antibody to aSMA (1:200), GDP and collagenase type 1 from Clostridium histolyticum were from Sigma-Aldrich; GTPgS was from Cytoskeleton, Inc.; the mouse monoclonal antibodies to Cav1 (1:200) and Cav3 (1:200) were from BD Biosciences; the rabbit polyclonal antibodies to MYPT1 (1:500), phospho-MYPT1 at Thr853 (pMYPT1Thr853, 1:500), and phospho-MLC2 at Ser19 (pMLC2Ser19, 1:500 for Western blotting, 1:50 for immunostaining) were from Cell Signaling Technology, Inc.; and the rabbit polyclonal antibody to Ki67 (1:100) and FITC-conjugated monoclonal antibody to aSMA (1:50) were from Abcam Biochemicals.

Techniques: Phospho-proteomics