MassIVE MSV000089108

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TARGETING LIPA INDEPENDENT OF ITS LIPASE ACTIVITY IS A THERAPEUTIC STRATEGY IN SOLID TUMORS VIA INDUCTION OF ENDOPLASMIC RETICULUM STRESS

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Description

pCW57-LIPA-TurboID transduced cells were treated with or without 1mg/mL doxycycline for 48h, then were treated with 50mM biotin for 15min and then harvested and lysed in RIPA buffer (Thermo Scientific) supplemented with Halt protease inhibitor cocktail (Thermo Scientific) and Phosphatase Inhibitor Cocktail set I and set II (MilliporeSigma) on ice for 15min, followed by 20min of 20,000g centrifuge at 4 degrees C. Cleared cell lysates were incubated with M-270 streptavidin Dynabeads overnight. Then the beads were washed twice with RIPA buffer (2min), once with 1M KCl (2min), once with 0.1M Na2CO3 (10s), once with 2M urea in 10mM Tris-HCl (pH 8.0) (10s), and twice with RIPA buffer (2min). Enriched material was eluted from beads by boiling samples in 4x Laemmli buffer supplemented with 2mM biotin and 20mM DTT at 95 degrees C for 10min. Eluted samples were resolved in SDS-PAGE and followed by mass spectrometry by Lumos. biological replicate 1, 929193 (clone 1, no Dox.), 929194 (clone 1, with Dox.), 929196 (clone 2, no Dox.) and 929197 (clone 2, with Dox.). biological replicate 2, 932804 (clone 1, no Dox.), 932805 (clone 1, with Dox.), 932807 (clone 2, no Dox.) and 932808 (clone 2, with Dox.). [doi:10.25345/C5Q23R40R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: LIPA ; Turbo-ID

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Principal Investigators:
(in alphabetical order) 		Ganesh Raj, University of Texas Southwestern Medical Center, USA
Submitting User: jjotto
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