Display Settings:

Items per page

Results: 7

1.
FIGURE 7.

FIGURE 7. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

DNA methylation in the proximal promoter of CHRNA7. DNA isolated from SH-SY5Y, SK-N-BE, HeLa, and SH-EP1 cells was used for sodium bisulfite conversion to identify methylated CpG islands within the CHRNA7 promoter region. The proximal promoter region is indicated by the location of the CpG islands and the percentage of samples showing methylation. SH-EP1 cells show heavy methylation over the region where the original AP-2α binding site is located (−71).

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
2.
FIGURE 4.

FIGURE 4. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

Knockdown of AP-2α and effects on endogenous CHRNA7 mRNA levels. Multiple cell lines were transfected with 50 pmol of siRNA (random or AP-2α-specific) for 48 h. A, total cell lysates were used in Western blot analysis to determine AP-2α protein levels using specific antibody. GAPDH served as a loading control. B, RNA was extracted, and cDNA was made. Levels of AP-2α and CHRNA7 mRNA were measured using quantitative PCR and normalized to GAPDH levels. Triplicates were used for each experiment, and experiments were replicated in triplicate. Protein levels from Fig. 4A were quantified using ImageJ (National Institutes of Health) and normalized to GAPDH protein levels. Data are presented as mean ± S.E. *, p < 0.05; **, p < 0.0005.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
3.
FIGURE 1.

FIGURE 1. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

Inverse correlation between AP-2α and CHRNA7 mRNA expression. A, sequence of the proximal promoter of the CHRNA7, with putative Sp and Egr-1 sites indicated by black and gray bars, respectively, and the AP-2α binding site indicated by the red bar. Mutation of the AP-2α site used in this study is shown in red. B, using quantitative real-time PCR, RNA isolated from SH-SY5Y, SK-N-BE, HepG2, HeLa, SK-N-MC, and SH-EP1 cells grown in complete medium was reverse transcribed to cDNA and analyzed for the expression of Sp1, Sp3, Sp4, Egr-1, AP-2α, and CHRNA7 mRNA, which was normalized to GAPDH mRNA levels. Triplicates were used for each experiment, and experiments were replicated in triplicate. Data are presented as mean ± S.E. (error bars). Ø, undetectable cDNA level after 40 PCR cycles.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
4.
FIGURE 2.

FIGURE 2. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

Mutation analysis of a putative AP-2α binding site using a reporter gene assay. A 230-bp CHRNA7 proximal promoter fragment was isolated and inserted upstream of a luciferase reporter gene in the vector pGL3-Basic and named Pr0.23B, with or without the AP-2α site mutated by site-directed mutagenesis. Transient transfection was performed using the Pr0.23B vector, AP-2mut vector, or pGL3-Basic with the Renilla luciferase vector pRL-TK as a control for transfection efficiency in multiple cell lines. Activity was determined by taking the ratio of luciferase to Renilla activity, and Pr0.23B activity was set to 100% for each cell line. The empty vector pGL3-Basic shows low expression of the luciferase reporter gene. Eight wells were used for each experiment, and experiments were replicated in triplicate. Data are presented as mean ± S.E. (error bars). *, p < 0.005. MCS, most common sequence.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
5.
FIGURE 5.

FIGURE 5. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

EMSA of binding to the CHRNA7 promoter AP-2 binding site at −71. Double-stranded oligonucleotides containing the normal (wt) or the mutated (mt) AP-2 site and the flanking sequences as found in the human most common sequence CHRNA7 promoter were 3′-end-labeled with biotin and incubated with nuclear extracts prepared from SH-SY5Y, SK-N-BE, SK-N-MC, HeLa (not shown), HepG2 (not shown), or SH-EP1 (not shown) cell lines as indicated. Extract was present in lanes 2–6 of each gel, wild type labeled probe was used in lanes 2–4 and 6, and mutated labeled probe was used in lane 5. Unlabeled competitor wild type probe was present in 100-fold excess in lane 3, and unlabeled competitor mutated probe was present in 100-fold excess in lane 4. Anti-AP-2α antibody was used for supershifting in lane 6. Protein-DNA complexes are indicated by the arrows labeled AP-2α, and the antibody supershifted complexes are indicated by the arrows labeled SS. Protein complexes indicated by the arrows labeled with asterisks are unknown ternary complexes. EMSAs were replicated two times for each cell extract, and three separately prepared extracts were used for each cell line.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
6.
FIGURE 3.

FIGURE 3. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

AP-2α overexpression and effects on dual luciferase reporter gene assay and endogenous CHRNA7 mRNA expression. A, transient transfection of the Pr0.23B vector with either the empty expression vector SP(RSV)-NN, full-length AP-2α expression vector SP(RSV)-AP2, or the truncated AP-2α expression vector that lacks DNA binding (SP(RSV)-AP2ΔN278), along with the Renilla luciferase vector pRL-0 as a control for transfection efficiency. Activity was determined by taking the ratio of luciferase to Renilla activity, and activity of Pr0.23B with the empty vector was set to 100% for each cell line. Eight wells were used for each experiment, and experiments were replicated in triplicate. Data are presented as means ± S.E. (error bars). *, p < 0.005. B, transient transfection of the empty expression vector SP(RSV)-NN, full-length AP-2α expression vector SP(RSV)-AP2, or the truncated AP-2α expression vector that lacks DNA binding (SP(RSV)-AP2ΔN278) in multiple cell lines. RNA was extracted, cDNA was made, and levels of CHRNA7 mRNA were measured using quantitative PCR and normalized to GAPDH mRNA levels. The level of CHRNA7 mRNA from cells transfected with the empty expression vector was set to 100%. Triplicates were used for each experiment, and experiments were replicated in triplicate. Data are presented as mean ± S.E. *, p < 0.0005.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.
7.
FIGURE 6.

FIGURE 6. From: Transcriptional Repression of the ?7 Nicotinic Acetylcholine Receptor Subunit Gene (CHRNA7) by Activating Protein-2? (AP-2?).

Chromatin immunoprecipitation identification of AP-2α binding sites in the CHRNA7 promoter. A, SH-SY5Y, SK-N-BE, SK-N-MC, HeLa, HepG2, or SH-EP1 cells were formaldehyde-cross-linked and lysed, and lysates were sonicated to obtain DNA fragments 200–300 bp in length. Immunoprecipitation was performed using Sepharose beads and anti-AP-2α antibody or no antibody plus lysate. DNA fragments isolated by immunoprecipitation were used for quantitative PCR to amplify relevant CHRNA7 promoter sequences as indicated, using the primers shown in Table 1. Fold enrichment compared with no antibody control is indicated. Ø, no change from controls. Triplicates were used for each experiment, and experiments were replicated in triplicate. Data are presented as mean ± S.E. (error bars). B, a second region containing AP-2α binding was identified using chromatin immunoprecipitation. Two putative AP-2α consensus binding sites are indicated by boxes within the CHRNA7 promoter region. The positions of primers ChIP-315F and ChIP-315R are underlined. C, transient transfection of SH-SY5Y, SK-N-BE, and SH-EP1 cells with luciferase reporter vectors containing 5′ deletions of the CHRNA7 promoter at the second putative AP-2α binding region, normalized to Renilla expression from the pRL-TK vector. Eight wells were used for each experiment, and experiments were replicated in triplicate. −326 was set to 100% for each cell line. Data are presented as mean ± S.E. *, p < 0.0005.

Jessica Finlay-Schultz, et al. J Biol Chem. 2011 December 9;286(49):42123-42132.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk