Fluorescent conjugated secondary anti rabbit or anti mouse antibodies

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 were lysed directly with SDS sample buffer (4% sodium dodecyl sulfate [SDS], 20% glycerol, 50 mM Tris HCl (pH 6.8)), proteins separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were incubated overnight with specific primary antibodies diluted in blocking buffer. These included: TRAIL, PUMA, AKT, phospho-AKT(Ser473), phospho-p70S6K1(Thr389), ERKp44/p42, phospho-ERK(Thr202/Tyr204) and p-STAT1(Tyr701) from Cell Signaling Technology; MCL-1 and GAPDH from Santa Cruz Biotechnology; BCL-2 from DaKo; ISG15, ISG54, OAS1 and PML from Proteintech; β-actin from Millipore. After washing, bound antibodies were detected with fluorescent-conjugated secondary anti-rabbit or anti-mouse antibodies (Enzo Life Sciences), then visualized and quantified with an Odyssey system (Pierce, Waltham, MA, USA).