Pex14p | Peroxisomal marker
AS08 372 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Magnaporthe oryzae, Zea mays
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Twenty 5-day-old light grown Arabidopsis thaliana seedlings (wild type, the pex14-2 null allele, and the pex14-1 truncation allele) were ground with a pestle in a 1.5 mL tube on dry ice following the addition of a double volume (60 μL) of NuPAGE 2x loading buffer (Invitrogen). After centrifugation, 20 μL of supernatant was trasnferred to a fresh tube with 2.1 μL 0.5 M DTT and boiled at 100 °C for 5 mins. Samples were loaded onto NuPAGE 10% Bis-Tris gel (Invitrogen) next to Cruz Marker (Santa Cruz Biotechnology). After electrophoresis, proteins were transferred for 30 mins at 24 V to a Hybond ECL nitrocellulose membrane (Anersham Pharmacia Biotech) using NuPAGE transfer buffer (Invitrogen). The blot was blocked for 1 h 4 °C in 8% non-fat dry milk in TBS-T (blocking buffer),and incubated overnight wiht agitation at 4 °C with primary antibody (1:10 000 rabbit anti-PEX14, AS08 372, Agrisera) diluted in blocking buffer. The antibody soliution was decanted and the blot was rinsed 3 times, 5 min each, with blocking buffer at 4 °C. The blot was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000), diluted in blocking buffer for 4 h at 4 °C with agitation. The blot was washed with 3 times, 5 min each, with TBS-T and developed with chemiluminescent reagent (Advanta), 1:10 dilution in water.
Courtesy Sarah Burkhart and Bonnie Bartel, Rice University, USA.
Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 26377801
Journal: BMC Plant Biol
Figure Number: 5A
Published Date: 2015-09-16
First Author: Kao, Y. T. & Bartel, B.
Impact Factor: 4.142Open Publication
PEX5 is more membrane associated in pex4-1; high temperature does not rescue this defect. Extracts from dark-grown seedlings grown at 22 °C or 28 °C were cleared by a low-speed centrifugation to give homogenates (H). After a high-speed centrifugation, supernatants (S) were removed, and pellets were resuspended and spun at high speed to separate the wash supernatants (W) and final pellets (P). Equal volumes of each fraction were processed for immunoblotting by sequential incubation of the membrane with the indicated antibodies. PEX14 and mitochondrial (mito) ATP synthase are integral membrane proteins; HSC70 is mainly cytosolic. The positions of molecular mass markers (in kDa) are indicated on the left. Band intensities were quantified using ImageJ. For each sample, the supernatant, pellet, and wash were normalized to the corresponding homogenate to give the listed numbers
Pex14p has a protein transporter activity and is involved in protein targeting into peroxisome. Submitted protein name: Genomic DNA, chromosome 5, P1 clone:MQB2
Calero-Munoz et al. (2019). Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves. Plant Cell Environ. 2019 Sep;42(9):2696-2714. doi: 10.1111/pce.13597.
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Vincent et al. (2017). A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association. Plant J. 2017 Dec;92(6):1132-1142. doi: 10.1111/tpj.13749.
Zhong et al. (2016). MoDnm1 Dynamin Mediating Peroxisomal and Mitochondrial Fission in Complex with MoFis1 and MoMdv1 Is Important for Development of Functional Appressorium in Magnaporthe oryzae. PLoS Pathog. 2016 Aug 24;12(8):e1005823. doi: 10.1371/journal.ppat.1005823. eCollection 2016.
Li et al. (2015). Autophagic recycling plays a central role in maize nitrogen remobilization. Plant Cell. 2015 May;27(5):1389-408. doi: 10.1105/tpc.15.00158. Epub 2015 May 5.
Li et al. (2015). A Chaperone Function of NO CATALASE ACTIVITY1 Is Required to Maintain Catalase Activity and for Multiple Stress Responses in Arabidopsis. Plant Cell. 2015 Feb 19. pii: tpc.114.135095.
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