mlc pmlc serine 19  (Cell Signaling Technology Inc)

Journal: EMBO Molecular Medicine

Article Title: Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation

doi: 10.1002/emmm.201100192

Figure Lengend Snippet: Colonic and ileal paracellular permeability was determined with everted gut sac in STAT5 IEC WT and STAT5 IEC KO mice. Colonic and ileal mucosal RNA was extracted; long MLCK mRNA levels were quantitated by qPCR. Results were expressed as the mean ± SEM ( n = 10). * p < 0.05 versus STAT5 IEC WT mice. Colonic and ileal frozen sections were immunostained with pMLC (red). Negs are negative controls, n = 5. Ileal IECs were isolated from STAT5 IEC WT and STAT5 IEC KO mice, pMLC and MLC, ZO-1, -2 and -3, JAM-A, claudin-2 and occludin were identified with WB. Signal intensity was determined by densitometry. MLCK activation was expressed as the ratio of pMLC and MLC. n = 5. Ileal IECs were isolated using LCM, ZO-1, -2 and -3 mRNA levels were determined by qPCR, * p < 0.05 versus STAT5 IEC WT mice, n = 10. Ileal frozen sections were, respectively, immunostained with ZO-1 (green) or ZO-3 (red), n = 5. Insets are the confocal images. Original magnification, ×100, bar = 50 µm.

Article Snippet: Antibodies specific for MLC and phosphorylated MLC (serine 19) (pMLC), phospho-tyrosin694 specific STAT5 (pSTAT5), phospho-serine276 p65 (P-p65) antibodies were from Cell Signaling Technology (Danvers, MA).

Techniques: Permeability, Isolation, Activation Assay

Journal: EMBO Molecular Medicine

Article Title: Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation

doi: 10.1002/emmm.201100192

Figure Lengend Snippet: HT-29 IEC monolayers, grown on transwell filters, were interfered with stat5a , stat5b or stat5a and b ( stat5 ) siRNA. Paracellular permeability was assessed as apical-to-basolateral flux of FITC dextran (FD-4) clearance and TEER. Results were expressed as the mean ± SEM ( n = 7). TRANS: transfection reagent only. * p < 0.01 versus CON and TRANS. Total proteins (TP) were extracted from post-confluent HT-29 cell monolayer, ZO-1, -2 and -3, STAT5, cleaved Caspase-3, E-cadherin, pMLC and MLC were measured by WB ( n = 5). Signal intensity was determined by densitometry. * p < 0.01 versus TRANS.

Article Snippet: Antibodies specific for MLC and phosphorylated MLC (serine 19) (pMLC), phospho-tyrosin694 specific STAT5 (pSTAT5), phospho-serine276 p65 (P-p65) antibodies were from Cell Signaling Technology (Danvers, MA).

Techniques: Permeability, Transfection

Journal: EMBO Molecular Medicine

Article Title: Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation

doi: 10.1002/emmm.201100192

Figure Lengend Snippet: HT-29 IEC monolayers, grown on Transwell filters, were interfered with RelA / p65 and/or stat5a and b ( stat5 ) siRNA and exposed to IFN-γ (10 ng/ml) induction for 18 h and then stimulation with TNF-α (10 ng/ml) for 12 h. Para-cellular permeability in monolayers was assessed as FD-4 clearance and TEER. Total proteins (TP) were extracted, P-p65, p65, pMLC, MLC and Bax were determined by WB. Results were expressed as the mean ± SEM ( n = 6). ∧ p < 0.01 versus Controls (transfection reagent only), * p < 0.05 versus cytokine-treated controls, & p < 0.05 versus STAT5 knockdown monolayer. # p < 0.01 versus cytokine-treated STAT5 knockdown monolayer. STAT5 signalling in the IEC barrier function and mucosal injury. STAT5⊣ MLCK → ZOs feedback mechanism stabilizes IEC TJ barrier function under basal conditions (1); STAT5⊣ NFκB → MLCK negative feedback loop regulates IEC barrier function under inflammatory conditions (2) and mucosal immune responses to gut injury (3). MA, membrane-associated TJPs.

Article Snippet: Antibodies specific for MLC and phosphorylated MLC (serine 19) (pMLC), phospho-tyrosin694 specific STAT5 (pSTAT5), phospho-serine276 p65 (P-p65) antibodies were from Cell Signaling Technology (Danvers, MA).

Techniques: Permeability, Transfection, Knockdown, Membrane