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Pentrtm1a

Manufactured by Thermo Fisher Scientific
Sourced in United States

The PENTRTM1A is a laboratory instrument designed for the analysis of various sample types. It features advanced technology to perform core functions required for scientific investigations. The detailed specifications and intended applications of this product are not available at this time.

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2 protocols using pentrtm1a

1

Rice Protoplast Cell Death Assay

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The rice protoplast cell death assay was performed as a per previously described method [56 (link)]. The open reading frame of full-length candidate effector genes was amplified from the M. oryzae P131 cDNA library with the primers (Table S2) and sub-cloned into the entry vector pENTRTM1A (A10462, Invitrogen, Waltham, MA, USA). Then, the gateway-compatible vector pIPKb002 was used for the expression of these candidate effector genes [57 (link)]. The stem and sheath of 14-day-old etiolated rice seedlings were cut into 1.5 mm strips and soaked in a protoplast isolation buffer for 3 h in the dark with gentle shaking (50 rpm/min). Then the recombinant vectors were transfected into rice protoplast, which was suspended in transfection buffer 1 to a concentration of 3.5 × 105 cells/mL. A firefly luciferase (LUC) reaction with the lysate was performed using the Luciferase Assay System (E1500, Promega, Madison, GA, USA), and the LUC activity was detected using a luminometer (F200, Tecan, Männedorf, ZRH, CH).
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2

Cloning and Mutagenesis of NLRP3 Proteins

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Constructs coding for Flag-NLRP3 and Flag-NLRP3-LRR were a kind gift from F. Martinon (Unil, Lausanne, Switzerland), VSV-SGT1 from V. Petrilli (CRCL, Lyon, France), Myc-BRCC3 from W. Harper (Harvard Medical School, Boston), pcDNA3.1-HA-CSNK1A1 from P. Wang (Tongji University, Shanghai); pcDNA3-N-HA-NEK7 was purchased from Addgene. Human NLRP3 (hNLRP3), mouse NLRP3 (mNLRP3), and truncation deletions were cloned in pENTRTM1A (Invitrogen). Mutations were performed using QuickChangeTM II kit (Agilent Technologies). Primers are listed in Supplemental Table 3. cDNAs were transferred in pInducer21 using recombination Gateway LR clonase Enzyme mix kit (Thermofisher)38 (link).
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