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Top 20 open access protocols

1

Identifying Rhomboid Protease Cleavage Sites

To determine the cleavage site of rhomboid substrates, HEK293T cells were transfected as described above, lysed in
RIPA buffer, and subjected to anti-FLAG immunopurification (Sigma). Eluent was spotted onto a sinapinic acid matrix and
analyzed by MALDI-TOF mass spectrometry on a standards-calibrated Voyager DE Instrument (AB SCIEX) as previously described
(Moin and Urban, 2012 (link)). Resultant spectra were analyzed and plotted in the R
environment with aid of the MALDIquant package (Gibb and Strimmer, 2012).
Gandhi S., Baker R.P., Cho S., Stanchev S., Strisovsky K, & Urban S. (2020). Designed parasite-selective rhomboid inhibitors block invasion and clear blood-stage malaria. Cell chemical biology, 27(11), 1410-1424.e6.
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2

Purification and Mass Spectrometry of Proteolytic Products

Full-length substrate and C-terminal cleavage products were purified from in vitro proteolysis assays by anti-Flag immunoaffinity isolation and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix as described previously19 (link),32 .
Baker R.P, & Urban S. (2015). Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature, 523(7558), 101-105.
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3

Purification and Mass Spectrometry of Proteolytic Products

Full-length substrate and C-terminal cleavage products were purified from in vitro proteolysis assays by anti-Flag immunoaffinity isolation and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix as described previously19 (link),32 .
Baker R.P, & Urban S. (2015). Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature, 523(7558), 101-105.
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4

Membrane Modulation of HEK293T Cells

HEK293T cells (ATCC, Manassas, USA), which are neuronal in origin (Shaw et al., 2002 (link)), were transfected using X-tremeGENE HP (Roche, Basel, Switzerland), washed 22 hours post-transfection, and incubated in serum-free media containing membrane-altering agents (0.004% lyso-phospholipids or 2.5mM methyl-β-cyclodextrin) or NSAIDs (0.5mM each except 0.1mM for sulindac sulfide, or as specified otherwise) for ~18–24 hours. Cells were lysed in Laemmli buffer, resolved on 4–20% Tris-Glycine SDS-PAGE gels, and subjected to quantitative western analysis using infrared fluorescence (LiCor Biosciences, Lincoln, USA). Cell viability was assessed using trypan blue exclusion. MALDI-TOF mass spectrometry was performed as described (Moin and Urban, 2012 (link)).
Urban S, & Moin S.M. (2014). A subset of membrane-altering agents and γ-secretase modulators provoke non-substrate cleavage by rhomboid proteases. Cell reports, 8(5), 1241-1247.
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1012 2025

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