Journal: The Journal of clinical endocrinology and metabolism

Article Title: Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

doi: 10.1210/jcem.83.5.4825

Figure Lengend Snippet: FIG. 1. Time-dependent changes in level of mRNA coding CYP21 in response to forskolin, AII, and K1. Cells were maintained as described for the times shown in the presence of forskolin (10 mmol/L), AII (10 nmol/L), or K1 (14 mmol/L). Medium was then removed, and cellular RNA was recovered and subjected to Northern analysis for CYP21 mRNA. Results were quantified directly by PhosphorImager analysis and normalized to levels of GAPDH mRNA determined in the same lane. Results shown are the combined data (mean 6 SE) from at least three independent experiments in each case. Data are expressed as levels relative to the control value at time zero, and significant dif- ferences from control are indicated (*, P , 0.05).

Article Snippet: The blots were then washed in 2 3 SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 3 SSC containing 0.1% SDS at room temperature twice for 30 min each time before direct radioimaging quantification of bound probe using a PhosphorImager (4-h exposure, GS250 PhosphorImager and Molecular Analyst V1.4 Software, Bio-Rad, Hercules, CA) and subsequent exposure to film (Hyperfilm, Amersham).

Techniques: Northern Blot, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

doi: 10.1210/jcem.83.5.4825

Figure Lengend Snippet: FIG. 3. Changes in the level of mRNA coding CYP21 in response to agonists alone or in combination. Cells were maintained as described for 20 h in the presence of AII (10 nmol/L), K1 (14 mmol/L), BAY K8644 (1 mmol/L), TPA (10 nmol/L), dbcAMP (1 mmol/L), and fors- kolin (10 mmol/L) alone or in the combinations shown. Medium was then removed, and cellular RNA was recovered and subjected to Northern analysis for CYP21 mRNA. Results were quantified directly by PhosphorImager analysis and normalized to levels of GAPDH mRNA determined in the same lane. Results shown are the combined data (mean 6 SE) from at least three independent experiments in each case. Data are expressed as levels relative to the control value at time zero, and significant differences from control are indicated (*, P , 0.05).

Article Snippet: The blots were then washed in 2 3 SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 3 SSC containing 0.1% SDS at room temperature twice for 30 min each time before direct radioimaging quantification of bound probe using a PhosphorImager (4-h exposure, GS250 PhosphorImager and Molecular Analyst V1.4 Software, Bio-Rad, Hercules, CA) and subsequent exposure to film (Hyperfilm, Amersham).

Techniques: Northern Blot, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

doi: 10.1210/jcem.83.5.4825

Figure Lengend Snippet: FIG. 2. Concentration-dependent changes in the level of mRNA cod- ing CYP21 in response to K1. Cells were maintained for 20 h in the presence of K1 at the doses shown. Medium was then removed, and cellular RNA was recovered and subjected to Northern analysis as described. Results were quantified directly by PhosphorImager and normalized to levels of GAPDH mRNA in the same lane. Results shown are the combined data (mean 6 SE) from three independent experiments in each case. Data are expressed as levels relative to the control value (4 mmol/L K1), and significant differences from the control are indicated (*, P , 0.05).

Article Snippet: The blots were then washed in 2 3 SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 3 SSC containing 0.1% SDS at room temperature twice for 30 min each time before direct radioimaging quantification of bound probe using a PhosphorImager (4-h exposure, GS250 PhosphorImager and Molecular Analyst V1.4 Software, Bio-Rad, Hercules, CA) and subsequent exposure to film (Hyperfilm, Amersham).

Techniques: Concentration Assay, Northern Blot, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells.

doi: 10.1210/jcem.83.5.4825

Figure Lengend Snippet: FIG. 5. Effects of cycloheximide on agonist-induced increases in CYP11A, CYP17, 3bHSDII, and CYP21 mRNA. Cells were main- tained as described for 20 h in the presence of AII (10 nmol/L), for- skolin (10 mmol/L), or dbcAMP (1 mmol/L), in the presence or absence of cycloheximide (CX; 35 mmol/L). Medium was then removed, and cellular RNA was recovered and subjected to Northern analysis, prob- ing sequentially for 3bHSDII, CYP17, CYP11A, and CYP21 mRNA. Results were quantified directly by PhosphorImager analysis and compared to levels of 28S RNA in the same lane. Results shown are from one of three similar experiments.

Article Snippet: The blots were then washed in 2 3 SSC containing 0.1% SDS at room temperature for 15 min, and in 0.1 3 SSC containing 0.1% SDS at room temperature twice for 30 min each time before direct radioimaging quantification of bound probe using a PhosphorImager (4-h exposure, GS250 PhosphorImager and Molecular Analyst V1.4 Software, Bio-Rad, Hercules, CA) and subsequent exposure to film (Hyperfilm, Amersham).

Techniques: Northern Blot