Immunoblot analysis was performed as previously described
[14 (link)]. Cell lysates were separated with SDS-PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking of nonspecific binding sites for 2 hours with 5% nonfat milk in PBS with 0.1% Tween-20, we incubated the membranes with 1:1,000 anti-HAND1 polyclonal antibody or 1:1,000 anti-HAND2 monoclonal antibody (Abcam, Cambridge, MA, USA); anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, USA; dilution 1:100,000) was used as protein loading control. The proteins were detected by using Enhanced Chemiluminescent detection reagent (Amersham, Piscataway, NJ, USA), according to the manufacturer’s instructions.
[14 (link)]. Cell lysates were separated with SDS-PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking of nonspecific binding sites for 2 hours with 5% nonfat milk in PBS with 0.1% Tween-20, we incubated the membranes with 1:1,000 anti-HAND1 polyclonal antibody or 1:1,000 anti-HAND2 monoclonal antibody (Abcam, Cambridge, MA, USA); anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, USA; dilution 1:100,000) was used as protein loading control. The proteins were detected by using Enhanced Chemiluminescent detection reagent (Amersham, Piscataway, NJ, USA), according to the manufacturer’s instructions.
Free full text: Click here
