Protein concentrations were determined using the Pierce BCA Assay (Thermo Scientific, Rockford, IL). Western blot was performed as previously described [15 (link)]. Membranes were immunoblotted overnight with primary mouse monoclonal anti-RASSF8 Ab (1:1000, Abcam, Cambridge, MA, Cat.# ab56921), anti-P53 (BD Biosciences, Cat.# 554294) and anti-P21 (BD Biosciences, Cat.# 556431), mouse monoclonal anti-P65 Ab (Santa Cruz, Cat.# sc-8008) and rabbit polyclonal anti-Cyclin D1 Ab (1:1000, Santa Cruz, Cat.# sc-753), rabbit polyclonal anti-P50 Ab (1:1000, Cell Signaling, Danvers, MA, Cat.# 3035) and rabbit anti-IκBα Ab, anti-p-65 (1:1000, Cell Signaling, Danvers, MA, Cat.# 9936 and 3033).
After immunoblotting, the membranes were washed 3 times with PBST and incubated for 1 hr with horseradish peroxidase-conjugated goat anti-mouse Ab (1:5000, Santa Cruz) or horseradish peroxidase-conjugated rabbit anti-rabbit Ab (1:5000, Santa Cruz). Immunoreactive bands were visualized with the SuperSignal West Dura Extended Substrate Kit (Thermo Scientific, Rockford, IL) and the densities of protein bands were quantified by Alpha Ease FCTM software (Version 3.1.2, Alpha Innotech Corp, San Leandro, CA).
After immunoblotting, the membranes were washed 3 times with PBST and incubated for 1 hr with horseradish peroxidase-conjugated goat anti-mouse Ab (1:5000, Santa Cruz) or horseradish peroxidase-conjugated rabbit anti-rabbit Ab (1:5000, Santa Cruz). Immunoreactive bands were visualized with the SuperSignal West Dura Extended Substrate Kit (Thermo Scientific, Rockford, IL) and the densities of protein bands were quantified by Alpha Ease FCTM software (Version 3.1.2, Alpha Innotech Corp, San Leandro, CA).
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