1. Volume 58, Issue 3, Pages 1067-1077 (September 2000)
Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells 
Alistair J. Ingram, Leighton James, Hao Ly, Kerri Thai, Lu Cai, James W. Scholey 
Kidney International 
Volume 58, Issue 3, Pages (September 2000)
DOI: /j x
Copyright © 2000 International Society of Nephrology Terms and Conditions

2. Figure 1
Mesangial cells (MCs) were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for the times indicated. After immunoprecipitation with p44/42 MAPK monoclonal antibody and protein sepharose A beads, pellets were suspended with Elk-1 fusion protein and were incubated for 30 minutes at 30°C. Recovered protein was electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific anti–Elk-1 antibody, and visualized with an HRP-conjugated secondary antibody. (A) Exposure of MCs to stretch for the indicated times did not change the total amount of p44/42 MAPK protein present. (B) Exposure of MCs to stretch for the indicated times led to an increase in p44/42 MAPK activity, as measured by phosphorylation of a target Elk-1 protein, which was maximal at 10 minutes. (C) Densitometric data of experiments (N = 3 for each time point). Standard error bars are shown. All time points are significantly different from control (time 0) by pairwise comparisons using Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

3. Figure 2
Mesangial cells (MCs) were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for the times indicated. Isolated protein was then incubated overnight with c-Jun fusion protein beads. Recovered pellets were reacted for 30 minutes at 30°C for the kinase assay, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific c-Jun antibody, and visualized with an HRP-conjugated secondary antibody. (A) Exposure of MCs to stretch for the indicated times did not change the total amount of SAPK protein present. (B) Exposure of MCs to stretch for the indicated times led to an increase in SAPK activity, as measured by phosphorylation of a target c-Jun protein, which was maximal at 10 minutes. Both the major (33 kD) and minor (35 kD) isoforms are visible. (C) Densitometric data of experiments (N = 3 for each time point). Standard error bars are shown. All time points are significantly different from control (time 0) by pairwise comparisons using Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

4. Figure 3
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz), while incubated in 70 μmol/L SNAP for the times indicated. After immunoprecipitation with p44/42 MAPK monoclonal antibody and protein sepharose A beads, pellets were suspended with Elk-1 fusion protein and were incubated for 30 minutes at 30°C. Recovered protein was electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific anti–Elk-1 antibody, and visualized with an HRP-conjugated secondary antibody. (A) Neither exposure of MCs to stretch for the indicated times nor the presence of SNAP changed the total amount of p44/42 MAPK protein present. (B) Exposure of MCs to stretch for the indicated times led to an increase in p44/42 MAPK activity, as measured by phosphorylation of a target Elk-1 protein, which was maximal at 10 minutes. This increase could be largely prevented by preincubation with SNAP. (C) Densitometric data of experiments with standard error bars (N = 3 for each time point). The 2-, 5-, and 10-minute time points were significantly inhibited by SNAP when compared pairwise to their respective controls by Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

5. Figure 4
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) while being incubated in 70 μmol/L SNAP for the times indicated. Isolated protein was then incubated overnight with c-Jun fusion protein beads. Recovered pellets were reacted for 30 minutes at 30°C for the kinase assay, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific c-Jun antibody, and visualized with an HRP-conjugated secondary antibody. (A) Neither exposure of MC to stretch for the indicated times nor the presence of SNAP changed the total amount of SAPK protein present. (B) Exposure of MCs to stretch for the indicated times led to an increase in SAPK activity, as measured by phosphorylation of a target c-Jun protein, which was maximal at 10 minutes. This increase could be largely prevented by preincubation with SNAP. (C) Densitometric data of experiments with standard error bars (N = 3 for each time point). The 10-minute time point was significantly inhibited by SNAP when compared pairwise to the 10-minute stretch control by Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

6. Figure 5
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) while being incubated in 1 mmol/L 8-bromo-cGMP for the times indicated. After immunoprecipitation with p44/42 MAPK monoclonal antibody and protein sepharose A beads, pellets were suspended with Elk-1 fusion protein and were incubated for 30 minutes at 30°C. Recovered protein was electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific anti–Elk-1 antibody, and visualized with an HRP-conjugated secondary antibody. (A) Neither exposure of MC to stretch for the indicated times nor the presence of 8-bromo-cGMP changed the total amount of p44/42 MAPK protein present. (B) Exposure of MC to stretch for the indicated times led to an increase in p44/42 MAPK activity, as measured by phosphorylation of a target Elk-1 protein, which was maximal at 10 minutes. This increase could be largely prevented by preincubation with 8-bromo-cGMP. (C) Densitometric data of experiments with standard error bars (N = 3 for each time point). The 10-minute time point was significantly inhibited by 8-bromo-cGMP when compared pairwise to the 10-minute stretch control by Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

7. Figure 6
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) while being incubated in 1 mmol/L 8-bromo-cGMP for the times indicated. Isolated protein was then incubated overnight with c-Jun fusion protein beads. Recovered pellets were reacted for 30 minutes at 30°C for the kinase assay, electrophoresed, blotted to a nitrocellulose membrane, probed overnight with a phospho-specific c-Jun antibody, and visualized with an HRP-conjugated secondary antibody. (A) Neither exposure of MC to stretch for the indicated times nor the presence of 8-bromo-cGMP changed the total amount of SAPK protein present. (B) Exposure of MC to stretch for the indicated times led to an increase in SAPK activity, as measured by phosphorylation of a target c-Jun protein, which was maximal at 10 minutes. This increase could be largely prevented by preincubation with 8-bromo-cGMP. (C) Densitometric data of experiments with standard error bars (N = 3 for each time point). The 10-minute time point was significantly inhibited by 8-bromo-cGMP when compared pairwise to the 10-minute stretch control by Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

8. Figure 7
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for 10 minutes. After each stretch protocol, cells were washed and permeabilized and incubated with anti-phospho-p44/42 MAPK for 90 minutes. Cells were then washed and incubated with a secondary donkey anti-mouse Texas Red-conjugated antibody for 60 minutes. Cells were then washed and mounted directly on a glass slide, and confocal laser scanning microscopy was performed using a Bio-Rad MRC-600 confocal microscope. (A) Unstretched cells show primarily cytoplasmic staining. (B) Ten minutes of cyclic stretch induces a marked nuclear translocation of the immunofluorescent label, indicating translocation of phosphorylated p44/42 MAPK. (C) Preincubation with cGMP prior to stretch markedly inhibits p44/42 MAPK nuclear translocation in response to cyclic stretch for 10 minutes. (D) Little fluorescence was observed in the absence of primary (anti-phospho p44/42 MAPK) antibody.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

9. Figure 8
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa, 60 Hz) for 10 minutes. After each stretch protocol, cells were washed and permeabilized and incubated with anti-phospho-SAPK for 90 minutes. Cells were then washed and incubated with a secondary donkey anti-mouse Texas Red-conjugated antibody for 60 minutes. Cells were then washed and mounted directly on a glass slide, and confocal laser scanning microscopy was performed using a Bio-Rad MRC-600 confocal microscope. (A) Unstretched cells show primarily cytoplasmic staining. (B) Ten minutes of cyclic stretch induces a marked nuclear translocation of the immunofluorescent label, indicating translocation of phosphorylated SAPK. (C) Preincubation with cGMP prior to stretch markedly inhibits SAPK nuclear translocation in response to cyclic stretch for 10 minutes. (D) Little fluorescence is observed in the absence of primary (anti-phospho SAPK) antibody.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

10. Figure 9
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa) for 10 minutes. AP-1 gel shift assay was performed with isolated nuclear protein. Nuclear proteins were incubated with poly(dI-dC). poly(dI.dC) and were then reacted with radiolabeled consensus AP-1 oligonucleotides, electrophoresed, and autoradiographed. Retardation of the label was seen in response to stretch. One mmol/L 8-bromo cGMP significantly inhibited retardation of the label, indicating less nuclear protein bound to AP-1 consensus sequences. No retardation of radiolabel was seen in the absence of nuclear protein (neg). Specificity was ensured with competition experiments using excess unlabeled AP-1 consensus oligonucleotide, which revealed no retardation of the label (right lane). Experiments were performed in quadruplicate.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions

11. Figure 10
Mesangial cells were exposed to cyclic mechanical strain (-27 kPa) for 2 and 24 hours. Protein was isolated, and 40 μg per lane run on 12% SDS-PAGE gel and transferred to a nitrocellulose membrane, and then incubated one hour with monoclonal anti-PCNA antibody, followed by a one-hour incubation with a peroxidase-labeled anti-mouse secondary antibody. (A) Increased PCNA expression is seen in response to strain at 4 and 24 hours. This is completely abrogated by addition of 1 mmol/L 8-bromo-cGMP. (B) Densitometric data of experiments with standard error bars (N = 3 for each time point). Both 4- and 24-hour time points were significantly inhibited by 8-bromo-cGMP when compared pairwise with their respective stretch controls by Student's t-test.
Kidney International  , DOI: ( /j x)
Copyright © 2000 International Society of Nephrology Terms and Conditions