Protease inhibitors cocktail

Tissue samples or cells were lysed in radio-immunoprecipitation assay lysis buffer containing 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH8) and a protease inhibitors cocktail (Promega; Fitchburg, WI, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were blocked using 5% skimmed milk (BD Biosciences) in Tris-buffered saline with Tween 20 and then probed with specific primary antibodies against the following proteins: SDF-1 (1: 1000, ab9797; Abcam, Cambridge, MA, USA); GFP (1: 1000, ab290; Abcam); CXCR4 (1: 1000, #2024; Cell Signaling, Danvers, MA, USA); signal transducer and activator of transcription 3 (STAT3; 1: 2000, #4904; Cell Signaling, Danvers, MA, USA); p-STAT3 (1: 2000, #9145; Cell Signaling); GAPDH (1: 10000, #5632–1; Epitomics, Burlingame, CA, USA). Membranes were then subsequently incubated with horseradish peroxidase-coupled anti-rabbit or anti-mouse antibodies (both 1: 2500; Cell Signaling). Specific bands were visualized using ECL Blotting Detection Reagents (Amersham Biosciences; Little Chalfont, Buckinghamshire, UK).

The total protein was extracted using RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with protease inhibitors cocktail (Promega, G6521), protein concentration was measured by BCA kit (Beyotime Biotechnology, P0010S). After that as a standard western blot protocol, proteins were loaded and separated by 12% SDS-PAGE gel electrophoresis and incubated with primary and HRP-tagged secondary antibodies to detect the signal (antibody list in supplementary file Table S2).