Mouse anti noxa

Protein extraction was done with TritonX-containing lysis buffer and protein content was determined using BCA assay (Pierce^TM BCA protein assay, Thermo Fisher Scientific). SDS–PAGE was carried out followed by semidry blotting. After blocking the membrane for 1 h with 5% milk, proteins were detected using the following antibodies: rabbit anti-MCL-1 (Enzo, ADI-AAP-240F), mouse anti-BCL-2 (Dako (Agilent), M088701-2), rabbit anti-BCL-x_L (CellSignaling, 2762 S), mouse anti-NOXA (Enzo, ALX-804-408), mouse anti-BAX (BD Bioscience, 610983), rabbit anti-BAK (Upstate/ Merck, 06–536), rabbit anti-BIM (CellSignaling, 3183 S) and rabbit anti-caspase-3 (CellSignaling, 9662 S), mouse anti-β-Actin (Sigma, A5441), mouse anti-GAPDH (BioTrend (Hy Test Ltd), 5G4-6C5) or mouse anti-Vinculin (Sigma/Merck, V9131-100UL). Quantification of protein expression was performed using ImageJ 3.1 software.

Western blot analysis was performed as previously described^{43 (link)} using the following antibodies: mouse anti-NOXA, rat anti-BMF, rabbit anti-MCL-1 (Enzo Life Science, Farmingdale, NY, USA), mouse anti-BCL-2, rabbit anti-BAK (BD Biosciences), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-BIM, mouse anti-PARP, rabbit-anti PUMA, rabbit-anti BCL-x_L (Cell Signaling, Beverly, MA, USA), mouse anti-GAPDH (HyTest, Turku, Finland), rabbit anti-H3K4me2 (Diagenode, Liège, Belgium), rabbit anti-acetylated histone H3 (Merck Millipore, Darmstadt, Germany) and mouse anti-histone H3 (Abcam) or mouse anti-β-Actin (Sigma, Germany). Goat anti-mouse, goat anti-rabbit and goat anti-rat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odysee Imaging System, LI-COR Biosciences, Bad Homburg, Germany) were used for detection. For detection of histone modifications cells were lysed using RIPA buffer supplemented with Pierce Nuclease (Thermo Fisher, Waltham, MA, USA). Representative blots of at least two independent experiments are shown.

Western blot analysis was performed as described previously [44 (link)] using the following antibodies: Mouse anti-cFLIP (Enzo Life Sciences, Lörrach, Germany), goat anti-cIAP1 (R&D Systems, Wiesbaden, Germany), rat anti-cIAP2 (Enzo Life Sciences), mouse anti-XIAP (BD Biosciences, Heidelberg, Germany), rabbit anti-NIK (Cell Signaling Technologies, Beverly, MA USA), mouse anti-p100/p52 (Merck Millipore, Darmstadt, Germany), rabbit anti-p62 (MBL International, Woburn, MA, USA), mouse anti-Noxa (Enzo Life Sciences), rabbit anti-Mcl-1 (Enzo Life Sciences), mouse anti-caspase-8 (Enzo Life Sciences), rabbit anti-caspase-3 (Cell Signaling Technologies), rabbit anti-caspase-9 (Cell Signaling Technologies), mouse anti-β-actin (Sigma-Aldrich), mouse anti-GAPDH (HyTest, Turku, Finland), mouse anti-tubulin (Calbiochem, Merck Millipore, Darmstadt, Germany). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for enhanced chemiluminescence detection (Amersham Bioscience, Freiburg, Germany). Alternatively, secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of two or three independent experiments.