By Gabriella Castoria, Ferdinando Auricchio
Steroid Receptors: equipment and Protocols offers a variety of recommendations which have been lately utilized to the research of steroid receptors, robust instruments for the development of our knowing of either the mechanisms regulating gene transcription and the swift signaling responses of tissues to indications. learn during this zone has generated a wealth of knowledge permitting the elucidation of steroid receptor mechanisms and enhancing the therapy of many endocrine issues, primarily cancers. Chapters disguise how you can study gene transcription, chromatin and proteomic differences, extra-nuclear signaling rules, improvement of mobilephone and animal versions, and training of recent antibodies. Written within the profitable Methods in Molecular Biology sequence structure, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, comfortably reproducible protocols and notes on troubleshooting and keeping off recognized pitfalls.
Authoritative and simply obtainable, Steroid Receptors: tools and Protocols deals an up-to-date view on various sleek tools that may optimistically give a contribution to bettering our wisdom at the integration of steroid receptors in unmarried or assorted functionally attached mobile contexts lower than numerous physiological and pathological stipulations, mainly tumors.
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Extra resources for Steroid Receptors: Methods and Protocols (Methods in Molecular Biology)
14. Digest the plasmid with SalI (20 U) for 4 h at 37 °C and purify. Thereafter, generate the plasmid pGL3-PmiR140-cre as follows. 15. PCR-amplify a cre gene fragment (1,092 bp) from plasmid Cre-IRES-PuroR using primers 5′-ttccatttcaggtgtcgtga-3′ and 5′-ggaactTCTAGACTAATCGCCATCTTCCAGCA- 3′, purify and double digest with NcoI and XbaI (20 U each) for 4 h at 37 °C and purify. 16. Double digest the plasmid pGL3-PmiR140 (10 μg) with NcoI and XbaI (20 U each) for 4 h at 37 °C. 17. Run the digest mixture on a 1 % agarose gel as described in step 2.
For a 6-well plate: (a) Dilute 4 μg plasmid DNA in 250 μl Opti-MEM (b) Dilute 10 μl lipofectamine 2000 in 250 μl Opti-MEM. (c) Incubate at room temperature for 5 min. (d) Combine diluted DNA and lipofectamine followed by gentle mixing. (e) Incubate for 20 min. (f) Add 500 μl mixture to cells. (g) Change the medium after 4–6 h. (h) Visualize transfection efficiency in 24 h through fluorescent microscopy (see Note 10). 4 Activation of Estrogen Signaling 1. Twenty-four hours after transfection, treat the cells with 10 nM estradiol (or vehicle control) by adding 1 μL of 10 μM estradiol stock solution per each mL of growth medium.
Run the T4-DNA polymerase-treated mixture along with a 1 kb ladder on a 1 % agarose gel at 100 V for 1 h. 3 kb) using the DNA gel extraction kit. 8. Digest pCAG-GFP plasmid (10 μg) with SpeI (20 U) for 4 h at 37 °C and purify. 9. Treat with T4 DNA polymerase and purify with the PCR purification kit. 10. Treat with alkaline phosphatase according to the manufacturer’s instructions and purify (see Note 6). 11. 1, at 4 °C overnight (see Note 7). 12. 1 to obtain the plasmid pCAG-PmiR140-mCherry-GFP (Fig.