Protein was isolated from E13.5 placentas and embryos by acetone
precipitation from RNA-isolation buffer (Buffer RLT or TRK) and resuspending in
SUTEB loading buffer (8M Urea, 1% SDS, 10mM EDTA, 10mM Tris-HCl, pH 6.8).
Protein lysates were run on 4–20% SDS-PAGE acrylamide gels and
transferred to PVDF membrane (Millipore). Immunoblots were probed with anti-Mcm2
(Epitomics/Abcam), anti-Mcm2(Cell Signaling Technology), anti-androgen receptor
(Epitomics/Abcam), anti-SMAD2/3(Cell Signaling), anti-p21(Santa Cruz),
anti-MCM4(Cell Signaling Technology), anti-actin (Sigma). Secondary antibodies
used included goat anti-rabbit-HRP (Cell Signaling) and goat anti-mouse-HRP
(Sigma). Crescendo ECL substrate(Millipore) was used and immunoblots digitally
scanned using a cDigit scanner. Quantification of immunoblots was performed
using ImageStudio software.
precipitation from RNA-isolation buffer (Buffer RLT or TRK) and resuspending in
SUTEB loading buffer (8M Urea, 1% SDS, 10mM EDTA, 10mM Tris-HCl, pH 6.8).
Protein lysates were run on 4–20% SDS-PAGE acrylamide gels and
transferred to PVDF membrane (Millipore). Immunoblots were probed with anti-Mcm2
(Epitomics/Abcam), anti-Mcm2(Cell Signaling Technology), anti-androgen receptor
(Epitomics/Abcam), anti-SMAD2/3(Cell Signaling), anti-p21(Santa Cruz),
anti-MCM4(Cell Signaling Technology), anti-actin (Sigma). Secondary antibodies
used included goat anti-rabbit-HRP (Cell Signaling) and goat anti-mouse-HRP
(Sigma). Crescendo ECL substrate(Millipore) was used and immunoblots digitally
scanned using a cDigit scanner. Quantification of immunoblots was performed
using ImageStudio software.