Mouse anti caspase 8

Western blot analysis was performed as described previously [52 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-NOXA, rat anti-BMF (Alexis Biochemicals, Grünberg, Germany), mouse anti-AKT, mouse anti-BCL-2, mouse anti-BAX, rabbit anti-BAK (BD Bioscience), rabbit anti-caspase-3, mouse anti-caspase-9, rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, (Cell Signaling, Beverly, MA), rabbit anti-MCL-1 (Stressgene Bioreagents, Victoria, BC). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA)) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.

BV6-mediated killing mechanism was analyzed in co-cultures of tumor and CIK cells. TE671 (TE) cells that were efficiently killed by combination therapy and grown in adherent cultures allowing a segregation of effector and target cells by several washing steps were used for analyses. Analyzed samples included TE and CIK cells alone, TE and CIK cells pre-incubated with 2.5 μMol BV6 for 4 h, TE cells co-cultured with CIK cells at an E:T ratio of 20:1 for 3 h, and TE cells pre-incubated with 2.5 μMol BV6 for 4 h followed by co-culture with CIK cells at an E:T ratio of 20:1 for 3 h. Co-cultures of TE and CIK cells were washed for the removal of CIK cells before TE cells were analyzed by western blot analysis. Western blot analysis was performed as described previously using the following antibodies: mouse anti-caspase-8 (1:1000; Alexis Biochemicals, Gruenberg, Germany), rabbit anti-Bid, rabbit anti-caspase-3, and rabbit anti-caspase-9 (1:1000; Cell Signaling, Beverly, MA, USA). Mouse anti-β-actin (1:10000; Sigma) was used as loading control (37 (link)). Goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany).

Western blot analysis was performed as described previously [28 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-Noxa, rat anti-Bmf (Alexis Biochemicals, Grünberg, Germany), mouse anti-Bcl-2, rabbit anti-Bcl-x_L, mouse anti-Bax, rabbit anti-Bak (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Bim, mouse anti-PARP (Cell Signaling, Beverly, MA), acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-Mcl-1 (Stressgene, Victoria, BC), rabbit histone H3 (Abcam, Cambridge, UK). Mouse anti-GAPDH (HyTest, Turku, Finland), mouse α-Tubulin (Calbiochem, Darmstadt, Germany) or mouse β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies. Enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odyssey Imaging System, LICOR Bioscience, Bad Homburg, Germany) were used for detection. Representative blots of at least two independent experiments are shown.