Supersignal west dura extended substrate kit

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).

Ten-day-old seedlings were harvested into a 1.5 mL microcentrifuge tube, and their fresh weight was determined. Next, 2:1 (v/w) 4 × SDS-PAGE sample buffer [65 (link)] was added, and the seedlings were homogenized with a microcentrifuge pestle. To ensure efficient solubilization of plant proteins, homogenates were passed through 2 cycles of freezing in liquid nitrogen andboiling for 5 min. Tubes were centrifuged for 10 min at 12,000× g at room temperature, and supernatant samples were resolved by SDS PAGE. Proteins were electroblotted onto nitrocellulose membranes. ABI4-eGFP and β-actin were detected using the primary antibodies anti-GFP (Abcam, ab1218, Cambridge, UK) and anti-β-actin (Sigma, A4700, Saint Louis, MO, USA), respectively, and secondary peroxidase-coupled anti-mouse IgG antibody (Sera Care 5450–0011). Membranes were incubated in reaction mixes prepared from with a highly sensitive SuperSignal West Dura extended substrate kit (Thermo scientific, Waltham, MA, USA), and chemiluminescent signals were recorded using ImageQuant RT ECL Imager (GE Healthcare, Chicago, IL, USA).

Cell lysate was prepared for western blotting as previously described [37 (link)]. Protein concentrations were determined using the Pierce BCA Assay (Thermo Scientific, Waltham, MA). Membranes were immunoblotted overnight with the following primary Abs: rabbit anti-human PTEN Ab, rabbit anti-human p85 Ab, rabbit anti-human phospho-p85 Ab, rabbit anti-human p110-alfa Ab, rabbit anti-human p110-beta Ab, rabbit anti-human p110 gamma Ab (1:1000, Cell Signaling, Danvers, MA), rabbit anti-human c-Met Ab (1:250, Life technologies), rabbit anti-human phosphor-c-Met Ab (p-c-Met), rabbit anti-human total-Akt Ab (1:500, Cell Signaling), rabbit anti-human phospho-Akt (p-Akt) Ab (Ser473, 1:100, Cell Signaling), mouse anti-human CDKN1A Ab (1:200, BD Pharmingen) and mouse anti-human β-actin Ab (loading control; 1:5000, Cell Signaling). After immunoblotting, the membranes were washed with 1X TBS containing 0.1% Tween-20, followed by a 1-hr incubation with horseradish peroxidase-conjugated rabbit anti-mouse Ab (1:5000, Santa Cruz Biotechnology) and goat anti-rabbit Ab (1:5000, Santa Cruz Biotechnology). Immunoreactive bands were visualized with the Super Signal West Dura Extended Substrate kit (Thermo Scientific) and the densities of the protein bands were quantified by the Alpha-Ease FCTM software (version 3.1.2, Alpha Innotech, San Leandro, CA).