Western blot analysis was performed as reported previously (24 (
link)). Cells were lysed in
radioimmunoprecipitation assay lysis buffer (EMD Millipore, Billerica, MA, USA). Total protein concentration was determined using a
Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (25 (
link)). Extracted protein (~30 µg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel, and was then transferred to a
polyvinylidene difluoride membrane (EMD Millipore Corporation, Billerica, MA, USA) at 80 V for 2 h at 4°C. The membrane was blocked in skim milk for 1 h, and subsequently incubated overnight at 4°C with a
mouse monoclonal anti-CXCR4 (Abcam; dilution 1:2,000; #ab58176) or a rabbit monoclonal anti-EGFR (Cell Signaling Technology, Inc., Danvers, MA, USA; dilution 1:3,000; #4405) antibody. The following day, the membrane was gently washed in Tris-buffered saline with 0.1% Tween-20 (TBST), and then incubated with goat anti-mouse (#ZB-2305) or goat anti-rabbit (#ZB-2301)
horseradish peroxidase-conjugated secondary antibodies (OriGene Technologies, Inc.; dilution 1:2,000) for 2 h at room temperature. Subsequent to washing in TBST, the immunocomplexes were detected using
ECL Plus reagent (Applygen Technologies, Inc., Beijing, China) (16 (
link),26 (
link)).
Li R.H., Huang W.H., Wu J.D., Du C.W, & Zhang G.J. (2016). EGFR expression is associated with cytoplasmic staining of CXCR4 and predicts poor prognosis in triple-negative breast carcinomas. Oncology Letters, 13(2), 695-703.