FEBS J 283(14):2577–2598Įarnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Salvesen GS, Hempel A, Coll NS (2016) Protease signaling in animal and plant-regulated cell death. Minina EA, Staal J, Alvarez VE et al (2020) Classification and nomenclature of metacaspases and paracaspases: no more confusion with caspases. Tsiatsiani L, Van Breusegem F, Gallois P et al (2011) Metacaspases. Rawlings ND, Barrett AJ, Thomas PD et al (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Uren AG, O’Rourke K, Aravind L et al (2000) Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. In theory, this method can serve to detect damage-induced alterations of any protein-of-interest in any organism for which antibodies or fusion proteins are available, and hence, will greatly aid the study of rapid damage-activated proteolysis in the future. It is important to (1) keep plant tissues at all times on liquid nitrogen prior to protein extraction, and (2) denature the protein lysate as fast as possible, as metacaspase activation ensues quasi immediately because of tissue damage inherent to protein extraction. Here, we describe a protein extraction method to detect activation of AtMCA4 by Western blot with antibodies against endogenous AtMCA4 and a PROPEP1-YFP fusion protein. AtMCA4 subsequently cleaves PROPEP1, the precursor pro-protein of the plant elicitor peptide 1 (Pep1). Previously, we found that physical damage, e.g., pinching with forceps or grinding on liquid nitrogen of plant tissues, activates Arabidopsis thaliana METACASPASE 4 (AtMCA4). pone.Metacaspases are cysteine proteases that are present in plants, protists, fungi, and bacteria. Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. WB_Sup_7days_for_Densitom.png -> Prepared image for densitometry analysis of supernatant samples WB_Lysa_7days_for_Densitom.png -> Prepared image for densitometry analysis of lysate samplesĢ0171219 WB_Sup_7days_for_Densitom.png -> Correlation plot of fluorescence and pixel intensity for supernatant samples JPG -> Raw western blot image for supernatant samplesĢ0171219 Fluor_Densito_Correlation_Lys.png -> Correlation plot of fluorescence and pixel intensity for lysate samples Supernatant samples were obtained by spinning 1 mL sample at 15000 × g for 10 min and transferring 100 μL from each well to the clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA), followed by fluorescence measurement.Ġ21115_20171219 Correlation_Fluore_vs_Densito_mCherry_50mL_7_days.xlsx -> data points for fluorescence measurements and pixel intensity obtained with FiJI (Fiji Is Just ImageJ).Ġ22615 WB SP mCHerry Lysate 3.JPG -> Raw western blot image for lysate samplesĠ22615 WB SP mCHerry Supernatant 3. Fluorescence was measured at excitation 575/9 nm and emission 608/20 nm. Then, 100 μL of each strain was transferred to a clear bottom 96-well plate (Corning Costar, Tewksbury, MA, USA) and fluorescence was measured using an Infinite® M200 PRO plate reader (Tecan, Männedorf, Switzerland). Image present in this dataset were obtained by following the steps described in the protocol dx.doi.org/10.17504/protocols.io.kfpctmn.įluorescence measurements were obtained by growing cc1690 in 50 mL TAP media on a rotary shaker, set to 150 rpm, under constant illumination (50 μmol photons/m 2s). The results present in this dataset were obtained from recombinant Chlamydomonas reinhardtii expressing the fluorescent protein mCherry in different constructs formats, aiming to recombinant protein secretion.
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