Whole cell lysates (10 μg per lane) were analyzed by Western blotting as previously described [43 (link)]. Primary antibodies used were purchased from: ProScience/Axxora, Lörrach, Germany (anti-TRAIL-R2); Cell Signaling, Schwalbach, Germany (HMGA2, p-Src, Src, p-Akt, Akt,); R&D systems, Minneapolis, MN, USA (E-cadherin); Sigma-Aldrich, St. Louis, MO, USA (β-actin).
Protein levels were additionally assessed using FACS analysis. Briefly, 5 × 104 cells were seeded in 96-well plates and non-permeabilized or fixed and permeabilized (1% formalin, 30 min, 4oC, washing, 0.1% Tween, 30 min, 4oC, 2x washing) cells were incubated for 30 min at 4oC with primary antibodies against CXCR4 (BD Bioscience, San Jose, CA, USA), EGFR (AnaSpec Inc, Fremont, CA, USA), TRAIL-R2 or TRAIL-R1 (HS201 and HS101 both kindly provided by Henning Walczak, Imperial College, London). After washing (PBS, 1% BSA), cells were incubated with APC-labeled secondary antibody (Jackson ImmunoResearch, Newmarket, UK) (30 min, 4oC). Analysis was performed with a FACS Calibur and the CellQuest program (BD Bioscience).
Protein levels were additionally assessed using FACS analysis. Briefly, 5 × 104 cells were seeded in 96-well plates and non-permeabilized or fixed and permeabilized (1% formalin, 30 min, 4oC, washing, 0.1% Tween, 30 min, 4oC, 2x washing) cells were incubated for 30 min at 4oC with primary antibodies against CXCR4 (BD Bioscience, San Jose, CA, USA), EGFR (AnaSpec Inc, Fremont, CA, USA), TRAIL-R2 or TRAIL-R1 (HS201 and HS101 both kindly provided by Henning Walczak, Imperial College, London). After washing (PBS, 1% BSA), cells were incubated with APC-labeled secondary antibody (Jackson ImmunoResearch, Newmarket, UK) (30 min, 4oC). Analysis was performed with a FACS Calibur and the CellQuest program (BD Bioscience).