Double color infrared laser imaging system

Proteins were separated in SDS-PAGE gels and the separated proteins were transferred to PVDF membranes. The membranes were then blocked using 5% non-fat milk in PBST buffer (1 × PBS + 0.1% Tween 20) (PBSTM) for 30 min at room temperature with 60 r.p.m. shaking. The appropriate antibodies were then added to the PBSTM. Antibodies used were: anti-GST (1:5000; #M20007; Abmart), anti-His (1:5000; #M30111; Abmart), Anti-Flag (1:5000; #F3165; Sigma-Aldrich), anti-GFP (1:5000; #M20004; Abmart), and anti-RFP (1:5000; #5f8; Chromotek). After addition of the antibodies, the membranes were incubated at room temperature for 2–4 hr or 4°C for overnight; then washed three times (5 min each) with PBST buffer. The membranes were then incubated with a goat anti-mouse (Odyssey, no. 926 – 32210; Li-Cor) or anti-rabbit (Odyssey, no. 926–32211; Li-Cor) IRDye 800CW antibody (Odyssey, no. 926 – 32210; Li-Cor) at a dilution of 1:10,000 in PBSTM at room temperature for 30 min with 60 r.p.m. shaking; and followed by three washes (5 min each) with PBST. The signals were detected by excitation at 700 and 800 nm using a double color infrared laser imaging system (Odyssey, LI-COR company).

A cell lysis kit (Bio-Rad laboratories, Hercules, CA) was used to extract protein from 50-mg samples according to the manufacturer's instructions. Samples were ground on ice for 10 minutes in buffer (246 μL of lysis buffer, 1.25 μL of phosphatase inhibitor, 0.25 μL of protease inhibitor, and 2.5 μL of phenylmethanesulfonyl fluoride (PMSF) and then centrifuged (at 14,000 rpm) at 4°C for 15 minutes. Equal amounts of supernatant protein (60 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. The Western blot membrane was blocked with blocking buffer (Li-cor, Lincoln, NB) for 2 hours and then incubated with anti-E-cadherin (1:500; Abcam), anti-ZO-1 (1:500; Abcam), anti-vimentin (1:500, Abcam), anti-fibronectin (1:500; Abcam), anti-VEGF (1:500; Abcam), or anti-HIF-1α (1:500; Abcam) antibodies overnight for 12 to 16 hours at 4°C. The membranes were incubated with secondary antibodies (Li-cor, Lincoln, NB) at a 1:10,000 dilution for 1 hour at room temperature in the dark. A double-color infrared laser imaging system (Odyssey, Li-cor) was used for detection.