SILAC-Based Proteomic Analysis of Selinexor

IU-TAB1 cells were cultured for at least five passages in SILAC media containing L-arginine and L-lysine (light), or ¹³C₆-arginine and ¹³C₆-lysine (heavy; Cambridge Isotope Laboratories). Cells were then treated with DMSO (“light” labeled) or selinexor (400 nmol/L; “heavy” labeled) for 6 hours. Subcellular fractions were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) and validated by Western blot using anti-tubulin (cytoplasmic marker) and anti-histone H3 (nuclear marker). Equal amounts of protein lysates were mixed together for either nuclear or cytoplasmic fraction. The combined proteins were subjected to tryptic digestion, followed by purification of digested peptides, basic RPLC fractionation, LC-MS/MS, and data analyses as described elsewhere (15 (link)).

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Conforti F., Zhang X., Rao G., De Pas T., Yonemori Y., Rodriguez J.A., McCutcheon J.N., Rahhal R., Alberobello A.T., Wang Y., Zhang Y.W., Guha U, & Giaccone G. (2017). Therapeutic Effects of XPO1 Inhibition in Thymic Epithelial Tumors. Cancer research, 77(20), 5614-5627.

Publication 2017

L-arginine L-lysine 13C6-arginine 13C6-lysine NE-PER Nuclear and Cytoplasmic Extraction Reagents

Arginine Cells Cytoplasmic Digestion Dmso Fractionation Histone h3 Isotope Light Lysine Ms ms Peptides Proteins Selinexor Subcellular fractions Tryptic Tubulin Western blot

Corresponding Organization :

Other organizations : Georgetown University Medical Center, Georgetown University, European Institute of Oncology, National Institutes of Health, Chiba University, University of the Basque Country

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Variable analysis

independent variables

Treatment with DMSO (control)
Treatment with selinexor (400 nmol/L)

dependent variables

Subcellular localization of proteins
Protein abundance changes in nuclear and cytoplasmic fractions

control variables

Culturing of IU-TAB1 cells in SILAC media for at least five passages
Mixing of equal amounts of protein lysates from DMSO-treated (light) and selinexor-treated (heavy) cells for either nuclear or cytoplasmic fraction
Tryptic digestion of combined proteins
Purification of digested peptides
Basic RPLC fractionation
LC-MS/MS analysis
Validation of subcellular fractions by Western blot using anti-tubulin (cytoplasmic marker) and anti-histone H3 (nuclear marker)

controls

Negative control: DMSO treatment
Positive control: Not explicitly mentioned

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