Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity

Published: 24 January 2023| Version 1 | DOI: 10.17632/mcsrg3cgvh.1
Contributors:
Shao-En Ong,

Description

Raw western blot imaging data files (16-bit PNG from ProteinSimple imager) from Golkowski et. al. "Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity" in Molecular Cell (2023). File names match Supplemental Figures SI 8, 9, and 11.

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Antibodies used for immunoblotting were anti-E-cadherin (24E10, Cell Signaling Technology, CST, Cat # 3195), anti-AXL (C89E7, CST, Cat # 8661), anti-Snail (C15D3, CST, Cat # 3879), anti-ZEB1 (E2G6Y, CST, Cat # 70512), anti-CD44 (E7K2Y, CST, Cat # 37259), anti-GAPDH HRP conjugate (D16H11, CST, Cat # 8884), anti-AAK1 (E8M3P, CST, Cat # 61527), anti-RALBP1 (D87H8, CST, Cat # 5739), anti-REPS1 (D6F4, CST, Cat # 6404), anti-CDK4 (D9G3E, CST Cat # 12790), anti-CDK6 (DCS83, CST, Cat # 3136), anti-CDK2 (78B2, CST, Cat # 2546), anti-Cyclin D1 (92G2, CST, Cat # 2978), and anti-Cyclin D3 (DCS22, CST, Cat # 2936). Cell lysis and immunoblotting experiments were performed using standard procedures. Briefly, cells were rinsed twice with ice-cold phosphate buffered saline (PBS), lysed in modified RIPA buffer V1 (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 (v/v), 0.25% Na-deoxycholate (w/v), 1 mM EDTA, 10 mM NaF, 5% glycerol (v/v), pH 7.8) supplemented with HALT protease inhibitor (100x, Thermo Fisher Scientific, Waltham, MA), and lysates clarified by centrifugation at 21,000 rcf for 20 minutes at 4°C. Protein concentration was quantified using the Piece 660 nm Protein Assay Reagent (Pierce, Rockford, IL). Lysates were mixed with NuPAGE LDS Sample Buffer (4X, Thermo Fisher Scientific) containing 50 mM DTT and heated for 5 min at 95°C. 20 µg of protein was separated on Bolt 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and electro-transferred onto nitrocellulose membranes. The buffer used for blocking and antibody incubation was 5% BSA in TBS-T (50 mM NaCl, 150 mM Tris-HCl, 1% Tween-20, pH = 7.8). Membranes were incubated with goat anti-rabbit HRP conjugate, and bands visualized using the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA) and the Fluor Chem E imaging system (Protein Simple, San Jose, CA).

Institutions

University of Washington

Categories

Western Blot

Funding

National Institute of General Medical Sciences

R01GM129090

National Institutes of Health

R03TR003308

National Institute of General Medical Sciences

R01GM086858

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