Ab130244

The A549/DDP cell line or tumor tissues were rinsed with PBS and lysed in a RIPA buffer with protease inhibitors and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA) for Western blots. The proteins were segregated from tumor samples or cell lines. Procedures for immunoblotting are described elsewhere [18 (link)]. Primary antibodies against IL-7 (Abcam, ab9732, 1 : 3000 dilution), IL-7R (Abcam, ab180521, 1 : 1000 dilution), PI3K (CST, 13666, 1 : 1000 dilution), p-PI3K p85 alpha (phospho Y607) (Abcam, ab182651, 1 : 1000 dilution), AKT (CST, 4691, 1 : 1000 dilution), p-AKT (CST, 4691, 1 : 2000 dilution), ABCG2 (Abcam, ab130244, 1 : 1000 dilution), and GAPDH (Abcam, ab181602, 1 : 10000 dilution), as well as HRP-conjugated goat anti-rabbit secondary antibodies (Abcam, ab7090, 1 : 10000 dilution) and HRP-conjugated goat anti-mouse secondary antibodies (Abcam, ab47827, 1 : 5000 dilution), were procured from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Denver, MA).

Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).

Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 (1:1000, MAB2018, R&D Systems, USA), OCT-4 (1:1000, MAB1759, R&D Systems, USA), Nanog (1:1000, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:1000, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:1000, MBS462020, MyBiosource, USA), CD15 (1:1000, MAB2155, R&D Systems, USA), NESTIN (1:1000, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:1000, ab130244, Abcam, USA), VEGF (1:1000, MAB293, R&D Systems, USA) and HIF1α (1:1000, MAB1536, R&D Systems, USA). Enhanced chemiluminescence was conducted for visualization.