Maize Ubiquitin Promoter Gateway Cloning

The Maize Ubiquitin promoter from pTCK303 [67 ] was cloned into pMDC32 Gateway vector substituting 2×35S promoter to generate pMDC32-pUBI destination vector by Hind Ⅲ and Kpn Ⅰ, and STF, GFP and GUS were cloned into pMDC32-pUBI destination vector by using the Gateway system (Invitrogen). To generate the UBI::GFP-STF vector, GFP and STF were cloned separately, with 18 bp overlapping sequence between 3’GFP and 5’STF to acquire the GFP-STF sequence cloned into pMDC32-pUBI destination vector by using the Gateway system (Invitrogen). Constructs were introduced into Agrobacterium tumefaciens by electroporation or the freezing transformation method. A. tumefaciences strain AGL1 was used for Brachypodium, switchgrass and rice transformation as previously described [9 (link), 68 (link), 69 (link)].

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Wang H., Niu L., Fu C., Meng Y., Sang D., Yin P., Wu J., Tang Y., Lu T., Wang Z.Y., Tadege M, & Lin H. (2017). Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis. PLoS Genetics, 13(3), e1006649.

Publication 2017

Gateway system

Agrobacterium tumefaciens Brachypodium Electroporation Maize Rice Strain Switchgrass Ubiquitin Vector

Corresponding Organization : Oklahoma State University

Other organizations : Noble Research Institute

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Protocol cited in 1 other protocol

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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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 previously^{19 (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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Purification and Mass Spectrometry of Proteolytic Products

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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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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Variable analysis

independent variables

Maize Ubiquitin promoter from pTCK303

dependent variables

Expression of GFP, STF, and GUS

control variables

2×35S promoter
Agrobacterium tumefaciens strain AGL1 used for transformations in Brachypodium, switchgrass, and rice

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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