Journal:

Article Title: Hyperosmotic Stress Induces Nuclear Factor-?B Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells

doi:

Figure Lengend Snippet: Hyperosmolarity induces p38 and p42/44 activation in HT-29 cells (A). Cells were incubated with hyperosmotic medium prepared by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium for the indicated time periods. p38 and p42/44 activation was determined using Western blotting using antibodies raised against the active, double-phosphorylated form of p38 and p42/44. This figure is representative of two separate experiments. Treatment of HT-29 cells with the selective p38 inhibitor SB203580 or selective p42/44 inhibitor PD98059 suppresses the hyperosmolarity-induced IL-8 response (B). Hyperosmolarity was achieved by the addition of mannitol (for a final concentration of 100 mmol/L) to isosmolar medium. In control wells, cells were incubated with isosmolar medium. Data are mean ± SEM of n = 12 wells from two separate experiments. *, P < 0.05; **, P < 0.01. Dotted bar, no mannitol; cross-hatched bars, mannitol.

Article Snippet: The membrane was probed with anti-phospho-p38 or anti-phospho-p42/44 MAP kinase antibody (Promega) and subsequently incubated with a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Boehringer).

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot

Journal:

Article Title: Activin A in the Regulation of Corneal Neovascularization and Vascular Endothelial Growth Factor Expression

doi:

Figure Lengend Snippet: Activin A up-regulates VEGF expression in corneal epithelial cells in vitro. Cells were treated overnight with recombinant activin A or the inhibitors as described in the Materials and Methods section and VEGF protein levels were measured in their supernatant. Bars represent the VEGF protein levels released in the supernatant after the various treatments (mean ± SD). Treatment with activin A, but not with the vehicle (control) up-regulated VEGF protein levels, whereas inhibition of p42/44 MAPK or p38 MAPK suppressed activin A-induced VEGF up-regulation.

Article Snippet: Corneal lysates, or recombinant phopsho-p42/44 MAPK standards (provided by the manufacturer), were subsequently incubated with a monoclonal antibody against the phosphorylated (activated) form of p42/44 MAPK (Assay Designs), immobilized on a microtiter 96-well plate, for 1 hour at room temperature on a plate shaker at 500 rpm.

Techniques: Expressing, In Vitro, Recombinant, Control, Inhibition

Journal:

Article Title: Activin A in the Regulation of Corneal Neovascularization and Vascular Endothelial Growth Factor Expression

doi:

Figure Lengend Snippet: Activin A up-regulates both p38 and p42/44 MAPK activity in corneal epithelial cells. Cells were treated with activin A and p38 (A) and p42/44 (B) MAPK activity was measured as described in the Materials and Methods section. Bars represent the 450-nm reading that corresponds to the p38 or p42/44 MAPK activity (mean ± SD). Treatment with activin A up-regulated both p38 and p42/44 MAPK activity.

Article Snippet: Corneal lysates, or recombinant phopsho-p42/44 MAPK standards (provided by the manufacturer), were subsequently incubated with a monoclonal antibody against the phosphorylated (activated) form of p42/44 MAPK (Assay Designs), immobilized on a microtiter 96-well plate, for 1 hour at room temperature on a plate shaker at 500 rpm.

Techniques: Activity Assay

Journal:

Article Title: Activin A in the Regulation of Corneal Neovascularization and Vascular Endothelial Growth Factor Expression

doi:

Figure Lengend Snippet: P42/44 (A) and p38 (B) kinase activity increases during the course of corneal neovascularization in our murine model. Animals were treated as described in the Materials and Methods section, sacrificed on day 0 (D0) or day 7 (D7) after the scraping, and the activities of p42/44 and p38 were measured in corneal lysates. Bars represent the 450-nm reading that corresponds to the p42/44 (A) or p38 (B) kinase activity, respectively (mean ± SD). P42/44 and p38 kinase activity increased during corneal neovascularization in the murine model paralleling the increases in both activin A and VEGF. Administration of recombinant activin A (ActA) increased even further the activity of p42/44 and p38 MAPK, whereas administration of a neutralizing antibody against activin A (AntiA) decreased the activation of both p42/44 and p38 MAPK. Administration of the isotype-matched control antibody did not have a significant effect on the amount of either p38 or p42/44 MAPK.

Article Snippet: Corneal lysates, or recombinant phopsho-p42/44 MAPK standards (provided by the manufacturer), were subsequently incubated with a monoclonal antibody against the phosphorylated (activated) form of p42/44 MAPK (Assay Designs), immobilized on a microtiter 96-well plate, for 1 hour at room temperature on a plate shaker at 500 rpm.

Techniques: Activity Assay, Recombinant, Activation Assay, Control