Protein Extraction and Immunoblotting for SPHK2 and STAT Signaling

Total proteins were extracted using ice-cold lysis buffer (1% NP-40, 1mM DTT, phosphatase inhibitor and protease inhibitor cocktail in PBS) followed by immunoblotting [57 (link),58 (link)]. Primary antibodies against SPHK2, pSTAT1 (Y701), STAT1, IRF9, pSTAT2 (Y690), and STAT2 were purchased from Cell Signalling Technologies, USA. Anti-phosphorylated SPHK1 (S225) was obtained from ThermoFisher Scientific, Waltham, Massachusetts, USA; anti-SPHK1 from Abcam, USA; and anti-GAPDH from Santa Cruz Biotechnology, Santa Cruz, CA, USA. All images were captured using the ChemiDocTM XRS+ molecular imager (Bio-Rad Laboratories, Hercules, CA, USA).

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Hii L.W., Chung F.F., Mai C.W., Yee Z.Y., Chan H.H., Raja V.J., Dephoure N.E., Pyne N.J., Pyne S, & Leong C.O. (2020). Sphingosine Kinase 1 Regulates the Survival of Breast Cancer Stem Cells and Non-stem Breast Cancer Cells by Suppression of STAT1. Cells, 9(4), 886.

Publication 2020

pSTAT1 (Y701), pSTAT2 (Y690), anti-SPHK1 anti-GAPDH ChemiDocTM XRS+ molecular imager

Antibodies Buffer Cold Gapdh Irf9 Np 40 Phosphatase Protease inhibitor Proteins Sphk2 Stat1 Stat2

Corresponding Organization : International Medical University

Other organizations : Centre International de Recherche sur le Cancer, Cornell University, University of Strathclyde

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Variable analysis

independent variables

Lysis buffer (1% NP-40, 1mM DTT, phosphatase inhibitor and protease inhibitor cocktail in PBS)

dependent variables

Protein expression and phosphorylation of SPHK2, pSTAT1 (Y701), STAT1, IRF9, pSTAT2 (Y690), and STAT2

control variables

GAPDH (used as a loading control)

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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