Protein Extraction and Analysis Protocol

Total cell lysates were obtained with NP-40 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris, pH 8.0), supplemented with protease inhibitor cocktail (cOmplete mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein samples were denatured with 2-mercaptoethanol at 95 °C for 5 min. The following antibodies were used for detection: anti-pALK Y1604 (Cell Signaling Technology), anti-ALK (Cell Signaling Technology), anti-pSTAT3Y705 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-pERK1/2 (Cell Signaling Technology), anti-ERK1/2 (Cell Signaling Technology), anti-pAkt Y473 (Cell Signaling Technology), anti-AKT, anti-human PARP (Santa Cruz Biotechnology), anti-HDAC8 (H-145;polyclonal; Santa Cruz, Santa Cruz, CA, USA), anti-p-mTOR (Ser2448; Upstate), anti-p-S6K1 (Thr412; Upstate), anti-MET (Cell Signaling Technology), anti-MYCN (Santa Cruz), anti-ac-SMC3 (provided by Prof. K Shirahige, University of Tokyo, Tokyo, Japan) [55 (link)], anti-HSC70 (Santa Cruz), anti-β-actin (clone AC-15; Sigma), anti-actinin (H-2; Santa Cruz) and anti-GAPDH (clone 6C5; Merck).

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Shen J., Najafi S., Stäble S., Fabian J., Koeneke E., Kolbinger F.R., Wrobel J.K., Meder B., Distel M., Heimburg T., Sippl W., Jung M., Peterziel H., Kranz D., Boutros M., Westermann F., Witt O, & Oehme I. (2018). A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment. Cell Death and Differentiation, 25(12), 2053-2070.

Publication 2018

cOmplete mini, PhosSTOP anti-pALK Y1604 anti-ALK anti-pSTAT3Y705 anti-STAT3 anti-pERK1/2 anti-ERK1/2 anti-MET anti-MYCN anti-HSC70 anti-β-actin (clone AC-15; anti-actinin (H-2; anti-GAPDH (clone 6C5;

Actin Actinin Antibodies Buffer Cell Clone Erk1 Gapdh Human Inhibitors Mercaptoethanol Mtor Mycn Np 40 Phosphatase Protease inhibitor Protein Smc3 Sodium chloride Stat3 Tris

Corresponding Organization :

Other organizations : German Cancer Research Center, Heidelberg University, St Anna Children's Hospital, Martin Luther University Halle-Wittenberg, University of Freiburg, Hopp Children's Cancer Center Heidelberg, National Center for Tumor Diseases, University Hospital Heidelberg

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Variable analysis

independent variables

NP-40 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 50 mM Tris, pH 8.0), supplemented with protease inhibitor cocktail (cOmplete mini, Roche) and phosphatase inhibitors (PhosSTOP, Roche)

dependent variables

Protein expression and post-translational modifications detected by the following antibodies: anti-pALK Y1604, anti-ALK, anti-pSTAT3Y705, anti-STAT3, anti-pERK1/2, anti-ERK1/2, anti-pAkt Y473, anti-AKT, anti-human PARP, anti-HDAC8, anti-p-mTOR (Ser2448), anti-p-S6K1 (Thr412), anti-MET, anti-MYCN, anti-ac-SMC3, anti-HSC70, anti-β-actin, anti-actinin, and anti-GAPDH

control variables

Protein denaturation with 2-mercaptoethanol at 95 °C for 5 min

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