Journal: Kidney international

Article Title: Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance.

doi: 10.1046/j.1523-1755.2003.00804.x

Figure Lengend Snippet: Fig. 2. Effect of PD98059 on the cyclosporine A (CsA)–induced increase in transepithelial electrical resistance (TER) across Madin-Darby canine kidney (MDCK) cell monolayers. Cells were grown to confluency on Costar Transwell filters and treated for a period of 72 hours. Cells were pretreated for 1 hour with 10 mol/L PD98059 ( ) and 4.2 mol/L CsA ( ), prior to treatment with both (). Results are ex- pressed as the change in TER (TER) com- pared to time-matched control filters and are given as mean SEM of five independent experiments, each performed in duplicate. A one-way analysis of variance (ANOVA) was carried out and multiple comparisons between control and treatment groups were made using the Dunnett post test. Multiple comparisons between different treatments were carried out using the Bonferroni post test. *P 0.05, **P 0.01,*** P 0.001.

Article Snippet: MDCK cells were filters, transferred to 1.5 mL Eppendorf tubes (Cam-seeded at a density of 1 105 cells/mL and were seen to bridgeshire, UK), and kept on ice.

Techniques: Control

Journal: Kidney international

Article Title: Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance.

doi: 10.1046/j.1523-1755.2003.00804.x

Figure Lengend Snippet: Fig. 3. Effect of U0126 on the cyclosporine A (CsA)–induced increase in transepithelial electrical resistance (TER) across Madin-Darby canine kidney (MDCK) cell monolayers. Cells were grown to confluency on Costar HTS- Transwell cell culture inserts and treated for a period of 72 hours. Cells were pretreated for 1 hour with 10 mol/L U0126 ( ) and 4.2 mol/L CsA ( ), prior to treatment with both (). TER was measured using REMS robotic sys- tem (World Precision Instruments). Results are expressed as the change in TER (TER) com- pared to time-matched control filters and are given as mean SEM of five independent ex- periments, each performed in duplicate. A one- way analysis of variance (ANOVA) was carried out and multiple comparisons between control and treatment groups were made using the Dunnett post test. Multiple comparisons be- tween different treatments were carried out using the Bonferroni post test. *P 0.05, **P 0.01, ***P 0.001.

Article Snippet: MDCK cells were filters, transferred to 1.5 mL Eppendorf tubes (Cam-seeded at a density of 1 105 cells/mL and were seen to bridgeshire, UK), and kept on ice.

Techniques: Cell Culture, Control

Journal: Kidney international

Article Title: Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance.

doi: 10.1046/j.1523-1755.2003.00804.x

Figure Lengend Snippet: Fig. 4. Effect of SB203580 on the cyclosporine A (CsA)–induced increase in transepithelial electrical resistance (TER) across Madin-Darby canine kidney (MDCK) cell monolayers. Cells were grown to confluency on Costar Transwell filters and treated for a period of 72 hours. Cells were pretreated for 1 hour with 10 mol/L SB- 203580 ( ) and 4.2 mol/L CsA ( ), prior to treatment with both (). Results are expressed as the change in TER (TER) compared to time-matched control filters and are given as mean SEM of five independent experi- ments, each performed in duplicate. A one- way analysis of variance (ANOVA) was car- ried out and multiple comparisons between control and treatment groups were made using the Dunnett post test. Multiple comparisons between different treatments were carried out using the Bonferroni post test. **P 0.01, ***P 0.001.

Article Snippet: MDCK cells were filters, transferred to 1.5 mL Eppendorf tubes (Cam-seeded at a density of 1 105 cells/mL and were seen to bridgeshire, UK), and kept on ice.

Techniques: Control

Journal: Kidney international

Article Title: Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance.

doi: 10.1046/j.1523-1755.2003.00804.x

Figure Lengend Snippet: Fig. 7. Western blot analysis of phosphorylated p38 (A) and whole Fig. 6. Western blot analysis of phosphorylated extracellular signal- cell p38 (B) expression in Madin-Darby canine kidney (MDCK) cells regulated kinase (ERK) 1/2 (A) and whole cell ERK 1/2 (B) expression following treatment with the mitogen-activated protein kinase (MAPK) in Madin-Darby canine kidney (MDCK) cells following treatment with inhibitors PD98059 (PD) and SB203580 (SB) in the presence or absence the MAPK inhibitors PD98059 (PD) and SB203580 (SB) in the presence of cyclosporine A (CsA). MDCK cells were grown to confluency on or absence of cyclosporine A (CsA). MDCK cells were grown to conflu- Costar Transwell filters and treated as described for 24 hours. Cells ency on Costar Transwell filters and treated as described for 24 hours. were pretreated for 1 hour with 10 mol/L PD98059 or 10 mol/L Cells were pretreated for 1 hour with 10 mol/L PD98059 or 10 mol/L SB203580, either alone or in combination prior to treatment with 4.2 SB203580, either alone or in combination prior to treatment with 4.2 mol/L CsA. Whole cell lysates were then made using radioimmunopre- mol/L CsA. Whole cell lysates were then made using radioimmunopre- cipitation (RIPA) lysis buffer. Cellular proteins were separated electro- cipitation assay (RIPA) lysis buffer. Cellular proteins were separated phoretically on sodium dodecyl sulfate (SDS) polyacrylamide gel and electrophoretically on sodium dodecyl sulfate (SDS) polyacrylamide transferred to nitrocellulose membrane. Expression of phospho p38 gel and transferred to nitrocellulose membrane. Expression of phospho (A) was identified using a polyclonal rabbit antiphospho p38 antibody ERK 1/2 (A) was identified using a polyclonal rabbit antiphospho ERK and an antirabbit horseradish peroxide–conjugated secondary antibody. 1/2 antibody and an antirabbit horseradish peroxide–conjugated second- The nitrocellulose was then stripped of the antibody and expression of ary antibody. The nitrocellulose was then stripped of the antibody and whole cell p38 (B) was identified using a polyclonal rabbit anti-p38 expression of whole cell ERK 1/2 (B) was identified using a polyclonal antibody and an antirabbit horseradish peroxide–conjugated secondary rabbit anti-ERK 1/2 antibody and an antirabbit horseradish peroxide– antibody. Control (lane 1); 4.2 mol/L CsA (lane 2); 10 mol/L PD conjugated secondary antibody. Control (lane 1); 4.2 mol/L CsA (lane (lane 3); 10 mol/L PD plus 4.2 mol/L CsA (lane 4); 10 mol/L SB 2); 10 mol/L PD (lane 3); 10 mol/L PD plus 4.2 mol/L CsA (lane (lane 5); 10 mol/L SB plus 4.2 mol/L CsA (lane 6); 10 mol/L PD 4); 10 mol/L SB (lane 5); 10 mol/L SB plus 4.2 mol/L CsA (lane 6); plus 10 mol/L SB (lane 7); and 10 mol/L PD plus 10 mol/L SB plus 10 mol/L PD plus 10 mol/L SB (lane 7); and 10 mol/L PD plus 10 4.2 mol/L CsA (lane 8). mol/L SB plus 4.2 mol/L CsA (lane 8).

Article Snippet: MDCK cells were filters, transferred to 1.5 mL Eppendorf tubes (Cam-seeded at a density of 1 105 cells/mL and were seen to bridgeshire, UK), and kept on ice.

Techniques: Western Blot, Expressing, Lysis, Membrane, Control