Cxcr7

RIPA Lysis Buffer (Beyotime, Shanghai, China) was used to lyse the protein from cells or tissue samples. Proteins were then electrophorized on a 10% or 8% SDS-PAGE gel. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA), blocked in 5% skim milk powder for 2 hours, and incubated with the diluted antibody overnight at 4°C. The primary antibodies used were as follows: AKT, p-AKT Ser473 (Cell Signaling Technology, Danvers, Mass, USA), ERK, p-ERKThr202/Tyr204, PDGF (Abcam, Cambridge, UK), GAPDH, E2F1, VEGF, and CXCR7 (Proteintech, China). The protein bands were detected using ImageLab software and displayed using photographic film.

Cells were treated with lysis buffer (PBS containing 1% Triton X-100, protease inhibitor cocktail, and 1 mmol/L phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Equal concentrations of protein were subjected to SDS-PAGE. Following transfer to a Immobilon-P Transfer Membrane, successive incubations with anti-CXCR-4 (1:500; Rabbit; Proteintech, USA), CXCR-7 (1:500; Rabbit; Proteintech, USA), p65 (0.5 µg/mL; Abcam, USA), p-p65 (0.5 µg/mL; Abcam, USA), and p-Iĸb (0.6 µg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies or anti-GAPDH antibody (Sangon Biotech) were performed, followed by corresponding horseradish peroxidase-conjugated secondary antibody (Sangon Biotech) incubation. The immunoreactive proteins were then detected using the ECL system (NCM Biotech, Suzhou, China). Bands were scanned using a densitometer (GS-700; Bio-Rad) and quantification was performed via Quantity One 4.6.3 software (Bio-Rad). Experiments were repeated at a minimum of three times.

After SUM149 and MDA-MB-231 cells were treated with SSA (0, 2.5, 5, or 10 μM) for 24 h, total cell protein lysates were extracted using RIPA lysis buffer that contained a protease inhibitor cocktail and a phosphatase inhibitor cocktail. Protein lysates (40 μg), determined by BCA analysis (Beyotime, Shanghai, China), were then subjected to Western blotting analysis. The primary antibodies used in analyses were as follows: CXCR4 (1:1,000, Novus, USA), CXCR7 (1:1,000, Proteintech, China), AKT (1:500, Cell Signaling Technology, USA), p-AKT (1:2,000, Cell Signaling Technology, USA), mammalian target of rapamycin (mTOR, 1:1,000, Proteintech, China), matrix metalloproteinase-9 (MMP9, 1:500, Cell Signaling Technology, USA), MMP2 (1:500, Cell Signaling Technology, USA), and caspase 3 (1:1,000, Proteintech, China). The second antibody was a goat anti-rabbit secondary antibody coupled to horseradish peroxidase (1:20,000, Proteintech, China).
To further investigate the effect of SSA on the expression of these proteins in vivo, lungs from each group were collected and subjected to Western blotting as described above.