Results: 5

1.
Fig. 4.

Fig. 4. From: DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis.

Demethylation of the Pp in a region-specific and developmentally regulated manner in the mouse epididymis. A, Schematic diagram of the Pp showing the location of 4 CpG sites with respect to the ARE. B, Percent of methylation level at each CpG site in the Pp determined from genomic DNA obtained from three segments of postnatal epididymides at the indicated postnatal time points. The data comes from at least 10 clones per time point.

Anjana Bhardwaj, et al. Mol Endocrinol. 2012 April;26(4):538-549.
2.
Fig. 5.

Fig. 5. From: DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis.

Methylation inhibits AR recruitment and Pp-dependent transcription. A, Luciferase analysis of epididymal cells transiently transfected with mock and methylated Pp construct, performed as described in the legend of Fig. 1B. Cells incubated with the synthetic testosterone R1881 (T) and cotransfected with AR expression vector are indicated. B, ChIP analysis of DC2 cells transiently transfected with mock and methylated Pp construct using AR and a negative control antiserum. The values shown are mean (±se) obtained by real-time PCR from three samples run in triplicate. An asterisk indicates statistically significant differences from the control (P ≤ 0.05).

Anjana Bhardwaj, et al. Mol Endocrinol. 2012 April;26(4):538-549.
3.
Fig. 3.

Fig. 3. From: DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis.

Developmental and region-specific pattern of Ar and Pp expression and AR occupancy at the Pp. A and D, ChIP analysis of AR occupancy in adult epididymides segments or postnatal whole epididymides at the indicated time points. The values shown are mean ± se, obtained by real-time PCR from at least three pooled samples run in triplicate from three different experiments. AR occupancy is relative to the corpus-cauda region, which was given a value of 1. B, C, E, and F, Real-time PCR analysis of total cellular RNA from adult epididymides segments or whole postnatal epididymides at the indicated postnatal time points. The values shown are average fold (±se) from at least four independent samples, quantified as described in the Fig. 1E legend (the lowest value in each panel is set to 1). G, Western blot analysis of AR protein level using whole-cell lysates from the epididymides of mice from the indicated postnatal time points. The values shown are average fold (±se) from three individual blots that were each normalized to a loading control (β-actin and β-tubulin). The relative level of AR at d 20 was set to 1; an asterisk indicates statistically significant (P ≤ 0.05) differences from d 20. Supplemental Fig. 7 shows a representative blot.

Anjana Bhardwaj, et al. Mol Endocrinol. 2012 April;26(4):538-549.
4.
Fig. 1.

Fig. 1. From: DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis.

Identification of AREs responsible for Pp-dependent transcription in epididymal cells. A, Schematic diagram of a wild-type (Wt) Pp construct and ARE-mutant derivatives (mAREs). All constructs harbor 0.3 or 0.6 kb of 5′-flanking sequence and have the Renilla luciferase gene downstream (not shown). B–D, Luciferase analysis performed using epididymal cells incubated with or without the synthetic testosterone R1881 (T) and transiently transfected with the constructs shown in A (100 ng) and a simian virus 40 promoter-driven firefly luciferase plasmid PGL3-E-V (50 ng), the latter of which serves as an internal control for normalization. Some cells were also cotransfected with an AR expression vector (100 ng). Shown are average values ± se from three experiments done in triplicate (values are relative to cells transfected with the empty Renilla luciferase expression vector, which was given a value of 1). E, Real-time PCR analysis of total cellular RNA from adult wild-type and SPARKI mice epididymides was done using at least four independent samples that were normalized to the level of L19 mRNA, which encodes a ribosomal protein. mRNA levels in control mice were given a value of 1. Average values ± se are shown. TSS, Transcription start site; m, mutant. An asterisk indicates statistically significant differences from the control (P ≤ 0.05).

Anjana Bhardwaj, et al. Mol Endocrinol. 2012 April;26(4):538-549.
5.
Fig. 2.

Fig. 2. From: DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis.

Identification of GATA sites crucial for Pp-dependent transcription in vitro and in vivo. A, Schematic of the wild-type (Wt) construct (also shown in Fig. 1A), deletion mutants lacking region A (72 nt), region B (39 nt), or region C (25 nt), and site-specific mutants that lacked GATA consensus site G1, G2, or G3. B and C, Transient transfection analysis performed and quantified as described in the Fig. 1 legend with the constructs indicated. D, Schematic of the wild-type Pp transgene (Pem-214) previously described (25) and a derivative harboring the 2-nt mutation indicated (Pem-270). E, RNase protection analysis of whole epididymis total cellular RNA (10 μg) from adult transgenic mice containing either the Pem-214 or Pem-270 transgene. The transgene-specific probe (probe A) contains bovine GH (BGH) 3′ UTR sequences. A band of the expected size (∼200 nt) was protected by epididymis RNA. A β-actin probe was included in all assays as a loading control (the protected band was ∼35 nt). F, Transgene expression from five Pem-270 and two Pem-214 transgenic mouse lines (average values ± se, an asterisk indicates P ≤ 0.05). The average mRNA signal in GATA mutant mice was set as 1. T, Synthetic testosterone analog.

Anjana Bhardwaj, et al. Mol Endocrinol. 2012 April;26(4):538-549.

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