Anti cxcr4

RAW264.7 cells were washed twice with cold phosphate-buffered saline (PBS, pH = 7.0), and lysed in radioimmunoprecipitation (RIPA) buffer [Cell Signaling Technology (CST), USA] supplemented with protease inhibitors (Roche, USA). The protein concentration of each sample was assayed using the bicinchoninic acid method (BCA kit) (Pierce, Rockford, IL, USA). Equal amounts of protein (60 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% gel. Then, the protein was blotted onto a NC membrane. After blocking with 5% non-fat milk in 20 mM of tris-buffered saline (TBS) with 0.1% Tween for 1 h at room temperature with shaking, they were incubated with the indicated primary antibodies at 4 °C overnight, followed by the appropriate fluorescent secondary antibodies (1:5000 dilution) for 2 h at room temperature. The immune-reactive proteins were detected using the Odyssey laser digital imaging system (Gene Company). Primary antibodies employed in this study included anti-GAPDH (1:1000, CST, USA), anti-p65 (1:1000, CST, USA), anti-Erk1/2 (1:1000, CST, USA), anti-JNK (1:1000, CST, USA), anti-p38 (1:1000, CST, USA), anti-p-p65 (1:1000, CST, USA), anti-p-Erk1/2 (1:1000, CST, USA), anti-p-JNK (1:1000, CST, USA), anti-p-p38 (1:1000, CST, USA) and anti-CXCR4 (1:1000, Thermo Fisher, USA).

Frozen skeletal muscle and rectal tissue sections were fixed in 4% paraformaldehyde, washed in phosphate-buffered saline (PBS), and permeabilized with 0.3% Triton X-100 in PBS. Nonspecific binding was blocked by incubating sections with 10% (w/v) bovine serum albumin (BSA; Dako Cytomation, Santa Clara, CA, USA) in PBS for 15 min. The slices were subsequently incubated with anti-GFP (Abcam, Cambridge, UK), anti-CXCL12 (Proteintech, Rosemont, IL, USA), and anti-CXCR4 (Novus Biologicals, Centennial, CO, USA) for 2 h at a concentration of 5.0, 1.0, or 1.0 µg/mL. Next, the sections were incubated with fluorescence-labeled secondary antibody; for anti-GFP, Alexa 488 (Thermo Fisher Scientific, Waltham, MA, USA) was used; and for anti-CXCL12 and anti-CXCR4, Alexa 594 was used. Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 30 min. After washing, the stained slices were mounted using the ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were taken using BZ-X700 (Keyence, Tokyo, Japan).

Monoclonal antibodies to NF-κB (#MAB5078), and phospho-specific p65-NF-κB (#MAB7226), MMP-9 (#MAB911), polyclonal caspase-3 (#AF835) were purchased from R&D Systems (Heidelberg, Germany). Monoclonal antibodies to anti-FAK (#610088), anti-phospho-FAK (#558540) were from Becton Dickinson (Heidelberg, Germany). Monoclonal antibodies to β-actin (#A4700), resveratrol, alginate, DAPI, Fluoromount were from Sigma-Aldrich (Taufkirchen, Germany). Monoclonal anti-E-cadherin (#sc-21791), anti-vimentin (#sc-53464), anti-slug (#sc-166476), anti-paxillin (#sc-365059) normal mouse IgG (#sc-2025) were from Santa Cruz Biotechnology (Dallas, Texas, United States). Monoclonal anti-β1-integrin (#14-0299-82) and anti-CXCR4 (#35-8800) were from Thermo Fisher Scientific (Langenselbold, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence were from Dianova (Hamburg, Germany), and alkaline phosphatase-linked antibodies for Western blotting were from EMD Millipore (Schwalbach, Germany). resveratrol was prepared in 100 mM stocks with ethanol and directly diluted in the cell culture medium for CRC cell treatment, without exceeding an ethanol concentration of 0.1% during the investigations.