Mammalian Expression Vectors for AR and its Variants

The mammalian expression vectors for HA-tagged ubiquitin (HA-Ub), wild-type AR (AR-WT), and AR variants V2, V4, V5, and V7 were described previously (Bohrer et al., 2013 (link); Huang et al., 2005 (link); Liu et al., 2008 (link)). The AR variant v567es was kindly provided by Dr. Donald Tindall at Mayo Clinic. HA-SPOP and Myc-SPOP were cloned into pCMV vector. AR mutants S646A, S647A, T648A, and T649A and prostate-cancer-associated mutants of SPOP were generated by site-specific mutagenesis (Stratagene). AR deletion mutants ΔEGSSS, ΔASSTT, 2Δ, and Myc-SPOP-ΔBTB were generated by KOD-Plus Mutagenesis Kit (Toyobo). Antibodies used were anti-AR (N20), anti-ERK2, anti-Myc tag (Santa Cruz Biotechnology), anti-SPOP (Abcam), and anti-HA (Covance). MG132, cycloheximide, and mibolerone were purchased from Sigma-Aldrich. The antiandrogen enzalutamide was kindly provided by Medivation.

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An J., Wang C., Deng Y., Yu L, & Huang H. (2014). Destruction of Full-Length Androgen Receptor by Wild-Type SPOP, but Not Prostate-Cancer-Associated Mutants. Cell reports, 6(4), 657-669.

Publication 2014

KOD-Plus Mutagenesis Kit anti-AR (N20), anti-ERK2 anti-Myc tag anti-SPOP anti-HA MG132 cycloheximide mibolerone

Antiandrogen Antibodies Cancer of prostate Cycloheximide Deletion Enzalutamide Erk2 Mammalian Mg132 Mibolerone Mutagenesis Site specific mutagenesis Ubiquitin Vector

Corresponding Organization :

Other organizations : Mayo Clinic, Fudan University, University of Minnesota

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Variable analysis

independent variables

HA-tagged ubiquitin (HA-Ub)
Wild-type AR (AR-WT)
AR variants V2, V4, V5, and V7
AR variant v567es
HA-SPOP
Myc-SPOP
AR mutants S646A, S647A, T648A, and T649A
Prostate-cancer-associated mutants of SPOP
AR deletion mutants ΔEGSSS, ΔASSTT, 2Δ, and Myc-SPOP-ΔBTB

dependent variables

Not explicitly mentioned

control variables

Antibodies used were anti-AR (N20), anti-ERK2, anti-Myc tag (Santa Cruz Biotechnology), anti-SPOP (Abcam), and anti-HA (Covance)
MG132, cycloheximide, and mibolerone were purchased from Sigma-Aldrich
The antiandrogen enzalutamide was kindly provided by Medivation

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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