Tubulin antibodies

ABC294640 was purchased from DC Chemicals (Shanghai, China). SKI-II (4-[[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol) was purchased from Tocris Bioscience (Ellisville, Mo). Cell permeable short-chain C6 ceramide was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). S1P was purchased from Cayman Chemical Co. (Ann Arbor, MI). 5-fluorouracil (5-FU) and cisplatin were purchased from Sigma (Shanghai, China). SP600125 and JNK inhibitor II (JNKi-II) were obtained from Selleck (Shanghai, China). Antibodies against phospho (p)-AKT (Ser 473), p-AKT (Thr 308), p-ribosomal protein S6 kinase 1 (S6K1) (Thr-389), p-JNK1/2 (Thr 183/Tyr 185) and p-c-Jun (Ser73) were purchased form Cell Signaling Tech (Denver MA). Anti-AKT1, SphK2, c-Jun and tubulin antibodies were obtained from Santa Cruz (Santa Cruz, CA).

Antibodies against PDCD6 were purchased from Proteintech. Antibodies against phospho-c-Raf, c-Raf, phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-STAT3, Ki67 and cleaved-Caspase3 were purchased from Cell Signaling Technology. Tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc.

Forty μg proteins and 60 μg of protein lysates from HMC-1 and mice tissues, respectively, were loaded onto either 7, 10, or 12% mini-protean TGX precast gels (BIO-RAD, Hercules, CA, USA) and allowed to run at 100 V for 1.5 h. The blots were transferred to Immobilon-P polyvinylidene fluoride membrane (Millipore, Massachusetts, MA, USA) for 1 h at 100 V. The membranes were then blocked with 5% BSA-TBS (w/v) for 1 h at room temperature and incubated overnight at 4 °C with either antibodies for IL-31, tryptase (Abcam, Cambridge, UK), PKC, phosphor-PKC, PKC, ERK, phosphor-ERK, JNK, phosphor-JNK, p38, phospho-p38, IKKβ, Phosphor-IKKβ, IKBα, phosphor-IKBα, NF-κB, or phosphor-NF-κB (Santa Cruz Biotechnology, Dallas TX, USA). To ensure equal protein loading, the membranes were stripped and reprobed with β-actin (Biosciences, San Diego, CA, USA) or tubulin antibodies (Santa Cruz Biotechnology). Blots were incubated in respective secondary antibodies (Santa Cruz Biotechnology, CA, USA) in 5% milk-TBS for 2 h at room temperature and then visualized on an ultraviolet detection Imaging System using an enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ, USA). Band intensity was measured using the Image J gel analysis software.