Horseradish peroxidase conjugated hrp anti rabbit antibodies

The breast cancer cells were lysed with RIPA buffer with protease inhibitors (Sigma-Aldrich). Protein quantification was carried out using a BCA protein assay kit (Promega). The primary antibodies used for western blot analysis were as follows: Rabbit anti-human Notch1 antibody (#sc6014; 1:500; Santa Cruz Biotechnology), anti-p65-nuclear factor-κB (NF-κB) antibody (#8242; 1:1,000), anti-p50-NF-κB antibody (#3035; 1:1,000) and rabbit anti-human β-actin antibody (#4967, 1:1,000) (all from Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (#e62238, 1:5,000; Santa Cruz Biotechnology) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were then blocked with 5% (5 g/100 ml) non-fat dry milk (Bio-Rad, Hercules, CA, USA) in Tris-buffered saline plus Tween (TBS-T) buffer for 2 h. The blots were immunostained with primary antibody at 4°C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). Protein levels were normalized to β-actin.

NSLCL cells treated with ESMC for 48 h were lysed with RIPA lysis buffer containing protease inhibitor cocktail and phosphates inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min, then lysis buffer was collected, and centrifuged at 12,000 g, 4°C for 10 min. The protein lysates were resolved by SDS-PAGE, and separated proteins were transferred to PVDF membranes and blocked with 5% skimmed milk for 2 h. The primary antibodies used for western blotting were rabbit anti-human FOXO3 antibody (1:1,000; Santa Cruz Biotechnology, Inc.) and rabbit anti-human β-actin antibody (1:1,000; Santa Cruz Biotechnology, Inc.). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) were used as the secondary antibodies. The blots were incubated with the respective antibodies overnight at 4°C under gently shaking. Finally, the proteins were detected by using horseradish peroxidase labeled secondary antibodies and an enhanced chemiluminescence detection system.

The primary antibodies were: rabbit anti-human HIF-1α antibody (#3434T, 1:1,000), PTEN antibody (#9559T, 1:1,000), rabbit anti-human phospho-AKT antibody (#9271T, 1:1,000), and rabbit anti-human β-actin antibody (#4970T, 1:1,000) (all from Cell Signaling Technology). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the secondary antibodies. The AKT inhibitor wortmannin was purchased from Sigma-Aldrich. The concentration used was 50 µM and the cells were treated for 12 h before further experiments. Cell lysates in 1X SDS loading buffer (60 mM Tris-HCl, pH 6.8; 2% SDS; 20% glycerol; 0.25% bromophenol blue; and 1.25% 2-mercaptoethanol) were incubated at 100°C for 10 min to facilitate sample loading for conventional western blot analysis. The relative protein levels were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).