Protease inhibitors complete mini

RCAECs (1 × 10⁵/well) were added into the 6-well plates, which were employed for western blotting. A radioimmunoprecipitation assay (RIPA) (Applygen, Beijing, China) involving protease inhibitors (complete mini, Roche) was utilized for extracting the proteins. The BCA assay (Applygen) was employed for estimating the levels of proteins. After protein isolation on SDS-PAGE, they were shifted to nitrocellulose membranes (Millipore). Membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and incubated overnight at 4°C in 5% milk/tris-buffered saline containing 0.1% Tween-20 (TBST) with the following primary antibodies: rabbit anti-p-ephrinB (1 : 1000, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Nck-2 (1 : 1000, Abcam), rabbit anti-p-FAK (1 : 500, Abcam), rabbit anti-VE-cadherin (1 : 1000, Abcam), rabbit anti-Integrinα 5 (1 : 1000, Abcam), and rabbit anti-GAPDH (1 : 2000, Cell Signaling Technology). The membranes were incubated with appropriate peroxidase-conjugated secondary antibodies at 1 : 2000 for 2 h at room temperature, followed by the visualization of immunoreactive proteins on the film using an ECL kit (Millipore, Temecula, CA, USA).

The BCCs and CSCs were treated with evodiamine for specified time and the cells were harvested in the lysis buffer containing Tris-Cl pH 7.4, 1% NP-40, 1% triton X-100, 0.25% sodium deoxycholate, 150 mM sodium chloride, 10% glycerol, protease inhibitors (complete mini, Roche, South San Francisco, CA, USA), and phosphatase inhibitors (1 mM Na₃VO₄, 1 mM NaF, and 20 mM β-glycerophosphate) and sonicated. The cleared lysates were resolved in SDS-polyacrylamide gel by electrophoresis and then transferred to a PVDF membrane (Bio-rad, Hercules, CA, USA). The membranes were blocked by 5% non-fat dry milk in a buffer containing 20 mM Tris pH 7.4, 150 mM NaCl and 0.1 % Tween-20, then probed with primary antibodies and secondary antibodies with horse radish peroxidase. The signals were detected Supersignal West Femto (Thermo, Waltham, MA, USA), or Novex (Invitrogen, Grand Island, NY, USA). All original raw western blotting images are seen in Figure S6. The Abs used are listed in Table S2.

Proteins were extracted and purified by the method of TRIZOL (Invitrogen), suspended in SDS 5% with protease inhibitors (complete mini, Roche, Indianapolis, IN, USA), and quantified by the Lowry method. Total proteins (30 µg) were subjected to vertical electrophoresis in acrylamide gels 12%, and then were transferred to nitrocellulose membranes using Trans-Blot-Turbo equipment/Transfer System (BioRad, Hercules, CA, USA). Membranes were incubated overnight at 4°C, blocking with low fat milk 5% in PBS 1X/Tween20 0.1%, and incubated 2 hr with antibodies (MEOX2) 1∶1500, (TWIST1) 1∶1500, (β-actin) 1∶3000, or (GAPDH) 1∶3000 (Santa Cruz Biotechnology, Dallas, Texas, USA). Membranes were incubated during 1 hr with a secondary antibody (anti mouse HRP/anti rabbit HRP) 1∶10000, at room temperature and revealed with Luminol 1 min at room temperature using C-Digit scanner (Li-cor, Lincon, Nebraska, USA) and Hypercassette and Films (Amersham, Chalfont, Buckinghamshire, England).