Journal: American Journal of Physiology-Gastrointestinal and Liver Physiology

Article Title: IGFBP-3 and IGFBP-5 production by human intestinal muscle: reciprocal regulation by endogenous TGF-β1

doi: 10.1152/ajpgi.1998.275.6.g1282

Figure Lengend Snippet: Fig. 5. IGFBP-4 production is constant during culture. Production of soluble IGFBP-4 by cultured human intestinal muscle cells remained constant during all phases of culture. IGFBP-4 was identified by Western blot analysis in medium conditioned by cells on days 3, 7, or 14 of culture and quantitated by image scanning densitometry. Values represent means 6 SE of 3 separate experiments.

Article Snippet: Densitometric analysis was performed using computerized densitometry and ImageQuant NT software (Molecular Dynamics).

Techniques: Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

doi: 10.1074/jbc.m004267200

Figure Lengend Snippet: FIG. 3. Effect of cisplatin and PD98059 on p53 protein level. A, cis- platin-induced dose dependence of p53 protein. After 24 h of exposure to the in- dicated doses of cisplatin, A2780 cells were lysed, and Western blot analysis of p53 protein was performed. Fold increase of protein level was determined by densi- tometry and calculated as the ratio of treated samples to the untreated sample. B, time course of cisplatin-dependent in- duction of p53 protein and its inhibition by PD98059. A2780 cells were pretreated for 1 h with or without 100 mM PD98059 or the solvent Me2SO and harvested at the indicated times after treatment with or without 10 mg/ml cisplatin. The cell extracts were subjected to Western blot analysis for detection of p53 protein. Fold increase of protein level was determined by densitometry and calculated as the ra- tio of treated samples to untreated sam- ples at each time point. PD, PD98059.

Article Snippet: The intensity of the bands was quantitated by densitometry (Personal Densitometer, Molecular Dynamics). p53 Half-life Studies—Cells were grown in 100-mm Petri dishes for 72 h to 70–80% confluency.

Techniques: Western Blot, Inhibition, Solvent

Journal: Journal of Biological Chemistry

Article Title: Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

doi: 10.1074/jbc.m004267200

Figure Lengend Snippet: FIG. 4. p53 half-life studies. A2780 cells were pretreated with or without 100 mM PD98059 for 1 h, followed by treatment with or without 10 mg/ml cisplatin for 8 h (closed circles, control; open circles, 100 mM PD98059; closed triangles, 10 mg/ml cisplatin; open triangles, PD98059 and cisplatin). Cells were then incubated with 20 mg/ml cycloheximide, and cells were harvested at the indicated times after initiation of cycloheximide treatment. Amounts of p53 protein were detected at each time point by densitometry of Western blot analysis and were normal- ized to the amount present at the 0-h time point (initiation of cyclohex- imide treatment). Results represent the mean of three experiments; error bars, S.E. PD, PD98059.

Article Snippet: The intensity of the bands was quantitated by densitometry (Personal Densitometer, Molecular Dynamics). p53 Half-life Studies—Cells were grown in 100-mm Petri dishes for 72 h to 70–80% confluency.

Techniques: Control, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

doi: 10.1074/jbc.m004267200

Figure Lengend Snippet: FIG. 5. Co-immunoprecipitation of p53 and ERK1/2 proteins. A2780 cells were pretreated with or without 100 mM PD98059 for 1 h, followed by treatment with or without 10 mg/ml cisplatin for 24 h. Cellular extracts were immunoprecipitated with antibodies to ERK1 and ERK2 that had been covalently coupled to protein A-Sepharose. The immunoprecipitated product was then subjected to Western blot analysis using antibodies to p53 protein (top gel) or antibodies to ERK1 and ERK2 (to control for differences in ERK1/2 protein levels; bottom gel). Whole cellular extracts were used as Western blot controls (far right lane). Negative controls included beads only and immunoprecipi- tation with an unrelated antibody (not shown). Fold increase of protein level was determined by densitometry and calculated as the ratio of treated samples to untreated samples. DMSO, Me2SO.

Article Snippet: The intensity of the bands was quantitated by densitometry (Personal Densitometer, Molecular Dynamics). p53 Half-life Studies—Cells were grown in 100-mm Petri dishes for 72 h to 70–80% confluency.

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

doi: 10.1074/jbc.m004267200

Figure Lengend Snippet: FIG. 7. Effect of cisplatin and PD98059 on phosphorylation of p53 protein at serine 15. A, A2780 cells were pretreated with or without 100 mM PD98059 (or with the solvent Me2SO (DMSO)) for 1 h, followed by treatment with or without 10 mg/ml cisplatin for 18 h. Cell extracts were subjected to Western blot analysis using antibodies to p53 protein or p53 protein phosphoryl- ated at serine 15. The relative amount of phosphorylated p53 was calculated as the percent of total p53 using densitometry. B, A2780 cells were pretreated with 20 mM of the calpain/proteosome inhibitor LLnL for 2 h, then treated and processed as described in A.

Article Snippet: The intensity of the bands was quantitated by densitometry (Personal Densitometer, Molecular Dynamics). p53 Half-life Studies—Cells were grown in 100-mm Petri dishes for 72 h to 70–80% confluency.

Techniques: Phospho-proteomics, Solvent, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Effect of Extracellular Signal-regulated Kinase on p53 Accumulation in Response to Cisplatin

doi: 10.1074/jbc.m004267200

Figure Lengend Snippet: FIG. 8. Effect of SB202190, wortmannin, and caffeine on cisplatin-induced phosphorylation of p53 protein at serine 15. A2780 cells were pretreated with one or more of the following:1 h with Me2SO (control), 1 h with 100 mM PD98059, 1 h with 10 mM SB202190, 30 min with 200 mM wortmannin, 15 min with 2.5 mM caffeine. Cisplatin (10 mg/ml) was then added to the cultures, and the cells were harvested after 18 h. A, cell extracts were subjected to Western blot analysis using antibodies to p53 protein or p53 protein phosphorylated at serine 15. B, the fold increase of total p53 protein (left), phospho-p53 (Ser-15) (middle), and the ratio of phospho-p53 (Ser-15) to total p53 (right) was determined by densitometry. In each case the level of protein was calculated as the ratio of treated samples to the untreated control sample.

Article Snippet: The intensity of the bands was quantitated by densitometry (Personal Densitometer, Molecular Dynamics). p53 Half-life Studies—Cells were grown in 100-mm Petri dishes for 72 h to 70–80% confluency.

Techniques: Phospho-proteomics, Control, Western Blot