Phospho cxcr4 s339 ab74012

Total protein was extracted from cells lysates using RIPA lysis buffer and loading buffer as previous described.17 Proteins were separated with SDS‐PAGE and incubated with specific antibodies. Protein bands were visualized and quantified with an Odyssey system (Pierce, Waltham, MA, USA). The antibodies used were: AKT, phospho‐AKT (Ser473), phospho‐p70S6K1 (Thr389), ERK, phospho‐ERK (Thr202/Tyr204), MEK1/2, phospho‐MEK1/2 (Ser271/221), IκBα, phospho‐IκBα (Ser32/36), STAT3, phospho‐STAT3 (Tyr705), caspase 9, caspase 8, and PARP, all obtained from Cell Signaling Technology (Danvers, MA, USA). BCL‐xL/S, MCL‐1, NF‐κB(p65), NF‐κB(RelB), and CXCR4(4G10) were bought from Santa Cruz Biotechnology (Dallas, TX, USA); Ac‐H3K9 obtained from Active Motif (Carlsbad, CA, USA); Phospho‐CXCR4 (S339) (ab74012) was purchased from Abcam (Cambridge, UK); β‐actin was obtained from Millipore (Burlington, MA, USA); The BCL‐2 antibody was purchased from Dako (Agilent Technologies, Santa Clara, CA, USA). Fluorescent‐conjugated secondary anti‐rabbit or anti‐mouse antibodies were purchased from Enzo life sciences.

Cells receiving various treatments and collected at various time points were subjected to Western blot analysis as previously described [32 (link)]. Primary antibodies against IL-24 (1:2000; Introgen Therapeutics, Houston, TX, USA), GRK6 (SC-100380; 1:1000; Santa Cruz Biotechnology, Inc., California, CA, USA), phospho-CXCR4^S339 (ab74012) and CXCR4 (ab2074) (1:1000; Abcam, Cambridge, MA, USA), phospho-CXCR4^S324/325 (CP 4251; 1:1000; ECM Biosciences LLC, Versailles, KY, USA), phospho-AKT^S473 (Cat. No. 4060), total AKT (Cat. No. 9272), phospho-PRAS40^T246 (Cat. No. 2997), total PRAS40 (Cat. No. 2691), phospho-mTOR^S2448 (Cat. No. 2971) and total mTOR (Cat. No. 2983) (1:1000; Cell Signaling Technology Inc; Beverly, MA, USA), CXCR7 (PA5-28739; 1: 1000; Thermo Scientific; Rockford, IL, USA); HIF-1α (ABE 279; 1:1000; Millipore), Beta actin (1:2000; Sigma Chemicals) were purchased and used as recommended by the manufacturers. Proteins were detected using appropriate secondary antibodies (Santa Cruz Biotechnology, Inc., and Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and an enhanced chemiluminescence kit (Thermo Scientific). Protein levels were detected using chemiluminescence imaging system (Syngene, Frederick, MD) and quantified using Image Quant (Syngene) software.