Goat anti rabbit sc 2054

All cell culture media, supplements and modifying enzymes were purchased from Invitrogen (Karlsruhe, Germany) if not otherwise indicated. Doxorubicin was from Biotrend Chemicals (Cologne, Germany), paclitaxel, and propidium iodide were obtained from Sigma-Aldrich (Taufkirchen, Germany). Gö6983, KU55933, SB203580, U0126 and Chk2 inhibitor II were from Merck (Darmstadt, Germany). The caspase inhibitors Z-VDVAD-FMK and Z-VAD-FMK were derived from R & D Systems (Wiesbaden, Germany). The following antibodies were used in this study: anti-HuR (sc-5261) and anti-Lamin B1 (sc-20682) from Santa Cruz (Heidelberg, Germany), anti-caspase-2 (#611022, BD Biosciences, Heidelberg, Germany), anti-caspase-3 (#9662), anti-caspase-7 (#9494) anti-PARP (#9542) and anti-Bid (#2002) antibodies were from Cell Signaling (Frankfurt, Germany), and anti-β-actin (#A2228) from Sigma-Aldrich. The goat anti-rabbit (sc-2054) and goat anti-mouse (sc-20559) HRP-linked antibodies were from Santa Cruz and Alexa Fluor 488 goat anti-mouse was from Life Technologies (Darmstadt, Germany). Radionucleotides were from Perkin Elmer (Rodgau, Germany) and the ECL system and Hyperfilms were from GE Healthcare (Freiburg, Germany).

The following antibodies were used for blotting: polyclonal goat anti-mouse κ LC (1050–01, SouthernBiotech); mouse anti-GAPDH (MAB374, Millipore); mouse anti-Hsc70 (B-6) (sc-7298, Santa Cruz Biotechnology), mouse anti-VCP/p97 (ab11433, abcam, Cambridge, MA), mouse anti-20S proteasome subunit alpha (C8) (PW8110, Biomol, Hamburg, Germany), rabbit anti-PSMC2 (HPA019238, Sigma-Aldrich), mouse anti-actin (A5441, Sigma-Aldrich), rabbit anti-PDIA6 (NBP157999, Novus Biologicals). HRP-conjugated antibodies were used at 1:10,000 dilution and include goat anti-rabbit (sc-2054), donkey anti-goat (sc-2020), and goat anti-mouse (sc-2031) all purchased from Santa Cruz Biotechnology. The rabbit anti-ERdj3 antibody was produced in our lab (Shen and Hendershot, 2005 (link)). IRdye^® secondary antibodies (LI-COR Biosciences, all 1:20,000): goat anti-mouse IgG (925–32210), goat anti-rabbit IgG (925–68071), and donkey anti-goat IgG (926–68074).

Immunoblotting was done essentially as described in [47 (link)] with the following modifications: a protease inhibitor cocktail (Roche, 1:25) was used and no additional dithiothreitol was added to the sample mix including loading dye. Aliquots of cell lysates (15μg) were separated by polyacrylamide gel electrophoresis (SERVA Gel neutral pH 7,4 Gradient, Serva, Germany) and electrotransferred onto polyvinylidene difluoride (PVDF) or nitrocellulose membranes (0,2μm pore size). The following proteins were detected by specific antibodies: BSP (A4217, Immundiagnostik, Bensheim), cleaved caspase 3 (#9661), cleaved caspase 8 (#9496), cleaved caspase 9 (#9501), cleaved caspase 7 (#9491), DDIT3 (#2895), cleaved PARP (#9541), c-FOS (#2250), CD44 (#3570), ID2 (#3431)(Cell signaling technologies, USA), ATF3 (sc188) and β-actin (sc1615, Santa Cruz, Heidelberg, Germany), which served as internal loading control. Horseradish peroxidase–conjugated secondary antibody included goat anti-rat (sc2006), donkey anti – goat (sc2020), goat anti - mouse (sc2055) and goat anti - rabbit (sc2054) (all from Santa Cruz, Heidelberg, Germany). Densitometric analysis was performed using the Quantity One Program (Biorad Laboratories GmbH, Munich, Germany).