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10 protocols using Lhcb1

The first and second arabidopsis leaves were sampled at 48 or 72 h post-infiltration with 2-OHM (10 µM) at 24 h for protein analysis. Protein extracts were prepared with 40 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% Triton X-100, and 1 × Roche Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN, USA), and then centrifuged at 10,000× g for 10 min at 4 °C. Aliquots of the supernatant were mixed with sample buffer (Tris-HCL, pH 6.8, 10% sodium dodecyl sulfate [SDS], 10 mM DTT, 20% glycerol, and 0.05% bromophenol blue). SDS-polyacrylamide gel electrophoresis (PAGE) and a blot assay were performed as described previously in [27 (link)]. Antibodies against Lhcb1, Lhcb4, RBCL, and ClpR1 were purchased from Agrisera (Vannas, Sweden).
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Western blotting was performed according to standard protocols. All antibodies (i.e., PsaD, PsbA, Lhcb1, PsaH, and PsaL) were purchased from Agrisera and were used at approximately 1/1000 dilution, followed by anti-rabbit HRP-conjugated secondary antibody. Blots were developed using Pierce ECL plus chemiluminescent substrate and exposed to X-ray film (RPI™, Research Products International, Corp). Silver staining was performed using Pierce Mass spectrometry compatible silver staining kit according to the manufacturer’s instructions.
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Total proteins from barley leaves were extracted as described previously (Pötter and Kloppstech, 1993 (link)). The protein concentrations were determined using Bradford reagent (BioRad Protein Assay, Hercules, CA, USA).
The proteins were separated by SDS-PAGE and transferred on to a nitrocellulose membrane. Proteins were stained with colloidal Coomassie G-250 (Dyballa and Metzger, 2009 ). For the immunological detection of proteins, polyclonal antibodies to HvPAP14 and HvPAP14i (BioGenes, Berlin, Germany), SAG12, RbcL, LHCB1, LHCB5, PSBO, and PsaA (all from Agrisera, Vännäs, Sweden) were used. Immunoreactive proteins were visualized with a peroxidase-coupled secondary antibody employing chemiluminescence (ECL Select Amersham, Pierce Therma Scientific Waltham, MA, USA; Ultra TMA-6 Lumigen, Southfield, MI, USA).
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Protein extracts were prepared from rosette leaves of Arabidopsis thaliana. A 10mg aliquot of leaf tissue was ground in liquid nitrogen and homogenized with 100 μl of sample buffer [50mM TRIS–HCl, pH 6.8, 2mM EDTA, 10% (w/v) glycerol, 2% SDS, and 6% 2-mercaptoethanol] was used to suspend the protein extracts. The protein samples were subjected to SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma–Aldrich). Antibodies against photosynthetic proteins, including Lhca3, Lhcb1, Lhcb2, Lhcb4, Lhcb5, CP43, D1, and PsaA (Agrisera, Sweden), were used for immunoblot analysis. Each protein was detected using an electrochemiluminescence (ECL) system (WESTSAVE, AbFRONTIER, Seoul, Korea) according to the manufacturer’s manual.
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Thylakoid proteins were extracted from the leaves in the presence of 10 mM NaF under dim light [59 (link)], and then were separated by SDS−PAGE (6% acrylamide stacking gel + 15% separation gel + 6 M urea) based on equal chlorophyll. Proteins from the gel were subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Immobilone, MilliPore, Darmstadt, Germany) using standard methods, and then were detected using specific antibodies including Lhca1, Lhca3, PsaD, CP43, D1, D2, Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5, and Lhcb6 (Agrisera, Umea, Sweden). Phosphorylation of thylakoid proteins was detected by anti−phosphothreonine antibody (Cell Signaling, Ipswich, MA, USA). The immunoblotting signals of proteins were detected using horseradish peroxidase−conjugated anti−rabbit antibody (Agrisera Comp., Umea, Sweden) and ECL reagents (GE Healthcare, Buckinghamshire, UK). Quantity one software (v4.4, Bio−Rad Comp., Hercules, CA, USA) was used to quantify signal amplitude.
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For separation of native thylakoid protein complexes, large pore blue native polyacrylamide gel electrophoresis (lpBN-PAGE) was performed as described by Jarvi et al. (2011) (link) with thylakoids solubilized in 1% (w/v) n-dodecyl-β-D-maltoside (DM) or 1% (w/v) digitonin (DIG). Additionally, thylakoid proteins were solubilized in Laemmli buffer (Laemmli, 1970 (link)) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing 15% (w/v) acrylamide and 6 M urea. For all gels, thylakoid samples were loaded on equal Chl basis.
For immunoblotting, proteins were transferred on PVDF membrane (Millipore) and recognized by specific antibodies for LHCB1 (1:5000, Agrisera), LHCB2 (1:5000, Agrisera), PsaB (1:3000, Agrisera), STN7 (1:1000, Agrisera), and CP47 (1:3000, gift from Prof. Roberto Barbato). Phosphorylated threonine residues were recognized with p-Thr antibody (1:3000, New England Biolabs) and phosphorylated LHCB1 (p-LHCB1, 1:10000, Agrisera) and LHCB2 (p-LHCB2, 1:10000, Agrisera). For detection, horseradish peroxidase-linked secondary antibody (Agrisera) and Amersham ECL Western blotting detection reagents (GE Healthcare) were used.
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Thylakoid membrane proteins were isolated as described by Fristedt et al. [71 (link)]. Isolated thylakoid membrane protein was separated by SDS-PAGE (5% acrylamide stacking gel + 15% separation gel + 6 M urea) [72 (link)]. Western blotting analysis was performed according to Chen et al. [73 (link)]. The primary antibodies (all raised in rabbits) including anti-Arabidopsis D1, D2, CP43, LHCb1, LHCb2, LHCb3, LHCb4, LHCb5, LHCb6, LHCa1, LHCa2, LHCa3, and LHCa4, and horseradish-peroxidase-conjugated secondary antibody were purchased from Agrisera (Umea, Sweden). The Western blotting signal was detected by a chemiluminescent detection system (ECL, GE Healthcare, Buckinghamshire, UK). The quantification of immunoblots was done with Quantity One software (Bio-Rad, Hercules, CA, United States).
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Thylakoid membranes were separated using SDS-PAGE. Equal quantities of Chl (2 μg) or protein (20 µg) were loaded onto each lane. To analyze and appraise quantitatively the polypeptide content in the thylakoids, immunoblotting was performed as described by Towbin and coworkers (Towbin et al., 1979 (link)). Proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. The membrane was incubated with polyclonal antibodies raised in rabbits. We have used the specific primary antibodies against PSI core (PsaA, PsaB, PsaC, PsaD, PsaG, and PsaH), Lhcb proteins (Lhcb1, Lhcb2, Lhcb4, Lhcb5, and Lhcbm5), PSII core (PsbA, PsbC, PsbD, PsbO, and PsbP) (all purchased from AGRISERA, Vännäs, Sweden, www.agrisera.com), and Lhca proteins (Lhca1, Lhca2, Lhca3, Lhca4, Lhca5, Lhca6, Lhca7, and Lhca9). Peptide tag antibodies for Lhca complexes were developed in our laboratory (Yadavalli et al., 2012 (link)). Subsequently, secondary antibodies ligated to horseradish peroxidase (HRP) were applied. We performed 2D-BN-SDS-Western blotting with respective antibodies to test the protein content changes due to salt treatment. After salt treatment, an anti-phosphothreonine antibody was used to reveal phosphorylated threonine residues in PSII proteins. Chemiluminescence reagents were used to develop the PVDF membrane. The images were recorded with a Bio-Rad CCD camera.
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Proteins were extracted and equal quantities of total protein analyzed by immunoblotting as described previously (Sugliani et al., 2016) . The following antibodies were used: AtpB (Agrisera; polyclonal,catalog numberAS08304), LHCA1 (Agrisera; polyclonal, catalog number AS01 005), LHCB1 (Agrisera; polyclonal, catalog number AS01 004), LHCB9 (Agrisera; polyclonal, catalog number AS15 3088), PetA (Agrisera; polyclonal, catalog number AS08 306), PsaA (Agrisera; polyclonal, catalog number AS04 042), PsbA (Agrisera; polyclonal, catalog number AS05 084, and RelA (1/2000 dilution, raised against E. coli RelA and kindly provided by M.Cashel).
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Proteins were extracted and equal quantities of total protein analyzed by immunoblotting as described previously (Sugliani et al., 2016) . The following antibodies were used: AtpB (Agrisera; polyclonal,catalog numberAS08304), LHCA1 (Agrisera; polyclonal, catalog number AS01 005), LHCB1 (Agrisera; polyclonal, catalog number AS01 004), LHCB9 (Agrisera; polyclonal, catalog number AS15 3088), PetA (Agrisera; polyclonal, catalog number AS08 306), PsaA (Agrisera; polyclonal, catalog number AS04 042), PsbA (Agrisera; polyclonal, catalog number AS05 084, and RelA (1/2000 dilution, raised against E. coli RelA and kindly provided by M.Cashel).
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