Anti cxcr4

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.

Western blot analysis was performed as already described [21 (link), 30 (link), 34 (link)]. Briefly, for protein detection, primary antibodies anti-β-Actin ((C4) #sc-47,778; Santa Cruz, Dallas, USA), anti-PCNA ((PC10) #sc-56; Santa Cruz, Dallas, USA), anti-AMT (#10633–1-AP; Proteintech Europe, Manchester, UK), anti-GCSH (#16726–1-AP; Proteintech Europe, Manchester, UK), anti-SGPL1 ((H-300) #sc-67,368; Santa Cruz, Dallas, USA), anti-Ezrin ((3C12) #sc-58,758; Santa Cruz, Dallas, USA), anti-CXCR4 (#11073–2-AP; Proteintech Europe, Manchester, UK) P-Cadherin (#13773–1-AP; Proteintech Europe, Manchester, UK) and Stathmin (#3352; Cell Signaling, Danvers, USA) were incubated overnight at 4 °C followed by labelling with a horseradish peroxidase (HPR)-conjugated secondary antibody (mouse #7076; rabbit #7074P2; Cell Signaling, Danvers, USA) for 1 h at room temperature. Finally, the protein signals were visualized with the Clarity™ Western ECL Chemiluminescent Substrate (Bio-Rad Laboratories Inc., USA). Stain free-images and β-actin were used as loading control. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 6.0.1 software (Bio-Rad, München, Germany). Protein detection was repeated at least three times with individually prepared cell lysates from independent passaged cells.

Protein was extracted from colon tissues or colon cancer cell lines with radioimmunoprecipitation assay (RIPA) buffer with a proteinase inhibitor. The Protein BCA Assay Kit (Bio-Rad) was used to measure the protein concentration in the lysate. 20 μg of protein were separated by SDS-PAGE (80 V, 2.5 h) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After that, the PVDF membranes were treated with a primary antibody (4°C, overnight). Followed by treatment with a peroxidase-conjugated secondary antibody, all the blots were visualized using ECL solutions and quantified. The following antibodies were used: anti-MMP-2 (1:500, Proteintech, Wuhan, Hubei, China), anti-MMP-9 (1:500, Proteintech), anti-CXCR-4 (1:2000, Proteintech), anti-vimentin (1:2000, Proteintech), anti-N-cadherin (1:2000, Proteintech), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-DLCK3 (1:1000, Boster Bio, CA, USA) and anti-GAPDH (1:1000, Abcam, Cambridge, UK).