Western Blot Analysis of Protein Signaling

Western blotting analysis of total protein in cell lysates from cultured rat and/or human CCA cell lines was carried out as previously described,17 (link),20 (link) with the following primary Abs purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): S1PR2 (H64); S1PR3 (H70); ERK1 (C16); ERK-2 (C-14); p-ERK (E-4); Akt1/2/3/ (H-136); and p-Akt1/2/3/ (ser473). S1PR1 (Ab23695) was purchased from Abcam (Cambridge, MA). Protein bands were normalized against β-actin and detected with mouse monoclonal anti-actin Ab (ACTN05) from Thermo Scientific (Wilmington, DE).

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Liu R., Zhao R., Zhou X., Liang X., Campbell D.J., Zhang X., Zhang L., Shi R., Wang G., Pandak W.M., Sirica A.E., Hylemon P.B, & Zhou H. (2014). Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2. Hepatology (Baltimore, Md.), 60(3), 908-918.

Publication 2014

S1PR2 (H64); S1PR3 (H70); ERK1 (C16); ERK-2 (C-14); p-ERK (E-4); Akt1/2/3/ (H-136); p-Akt1/2/3/ (ser473). S1PR1 (Ab23695)

Actin Akt1 Cell Cell lines Erk 2 Erk1 Human Mouse Protein Protein blotting analysis

Corresponding Organization : Wenzhou Medical University

Other organizations : China Pharmaceutical University, Nanjing Medical University, Jiangsu Province Hospital

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Variable analysis

independent variables

Rat and/or human CCA cell lines

dependent variables

Total protein expression levels
Protein band normalized against β-actin

control variables

Primary antibodies purchased from Santa Cruz Biotechnology, Inc. and Abcam
Mouse monoclonal anti-actin Ab (ACTN05) from Thermo Scientific

controls

Western blotting analysis protocol as previously described in references 17 and 20

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