Anti α tubulin t 5168 antibody

Plitidepsin (C₅₇H₈₇N₇O₁₅, MW:1109.6, CAS No. 137219-37-5, APL), [¹⁴C]-plitidepsin (1.73 GBq/mmol), and a fluorescent coumarinated plitidepsin derivative (plitidepsin-DMAC) were prepared by PharmaMar (Colmenar Viejo, Spain). Stock solutions (1 mg/ml in DMSO) were prepared and stored at −20 °C. Complete (protease) and PhosStop (phosphatase) inhibitor cocktails were purchased from Roche Diagnostics (Mannheim, Germany). Anti-phospho-JNK (Thr183/Tyr185), anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-phospho-p38 (Thr180/Tyr182) antibodies were purchased from Cell Signaling Technologies, Inc (Beverly, MA, USA). Anti-eEF1A and secondary HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-eEF1A2 (GTX102326) antibody was purchased from GeneTex (Irving, CA, USA). Anti-α-Tubulin (#T5168) antibody and subtilisin (EC 3.4.21.62) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). A plasmid encoding eEF1A2-GFP (RG210716) was purchased from Origene (Rockville, MD, USA). Chromatography media (DEAE FF 16/10, SP HiPrep 16/10 and Superdex 200 16/600 columns) were obtained from GE Healthcare (Buckinghamshire, UK). All other reagents were from Sigma (St Louis, MO, USA).

Antibodies against iNOS (ab 15323) were purchased from Abcam (Cambridge, UK). Anti-arginase-1 (BD610708) antibody was purchased from BD Bioscience (Madrid, Spain), and anti-α-tubulin (T-5168) antibody was from Sigma-Aldrich (St Louis, MO, USA).

Whole lysates, proteins extracted from SKOV-3/PAX were separated by 10-15 % SDS-PAGE according to their molecular weights. Each protein was transferred to nitrocellulose blotting membrane and blotted by appropriate antibodies for following molecules at 4 °C; Anti-cleaved caspase-9 (sc22182), -p-p38 (sc7973), -p-JNK (sc6254), -ERK (sc1647), and -p-ERK (sc7383) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase-8 (#9496), -cleaved caspase-3 (#9661), -PARP (#9542), -p-Raf-1 (#9421), -AKT (#9272), -p-AKT (S473) (#9271), -JNK (#3708), and -p38 (#9212) antibodies were obtained from Cell Signaling Technology (Danver, MA, USA). Anti-α-tubulin (T5168) antibody was purchased from Sigma (St. Louis, MO). Next day, the membranes were washed three times in phosphate buffered saline with 0.01 % tween-20 (PBST) and incubated at room temperature for 1 hour with secondary antibodies. The membranes were then washed with PBST three times and visualized the protein bands using an enhanced chemiluminescence detection system (DoGen-Bio, Seoul, Korea) and exposed to X-ray film (Agfa-Gevaert N.V., Mortsel, Belgium).