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11 protocols using AS05085

To analyse potential changes in polypeptide composition upon NPQ formation/relaxation, glycine–sodium dodecyl sulphate polyacrylamide gel electrophoresis (glycine–SDS-PAGE; Laemmli 1970 (link)) was used, as in Qin et al. (2015b (link)). Stacking and separation gels of 4 and 12% acrylamide/bis-acrylamide (29:1) mix, respectively, were employed. Samples were denatured with a lithium dodecyl sulphate sample buffer (Ikeuchi and Inoue 1988 ) at 40 °C for 10 min, and 10 μg Chl of chloroplasts was loaded per lane. After electrophoresis, gels were used for electroblotting onto nitrocellulose membrane (GE Healthcare, UK) and the proteins were incubated overnight at 4 °C with antibodies raised against PsbS (Agrisera AS09533, 1:2000), LHCSR3 (Agrisera AS142766, 1:1000) and LHCSR1 (Agrisera AS142819, 1:1000). The β subunit of ATP synthase (ATP-B, Agrisera AS05085, 1:2000) was used as loading control. Enhanced chemiluminescence (WSE-6200HLuminiGraph II chemiluminescent imaging system, ATTO, Japan) was used to visualise protein bands after incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Agrisera AS09602, 1:20000).
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Total cell samples (2.5 x 106 parasites per lane) were resuspended in Laemmli buffer (2% (w/v) SDS, 125 mm Tris–HCl pH 6.8, 10% (w/v) glycerol, 0.04% (v/v) β-mercaptoethanol, and 0.002% (w/v) bromophenol blue) and boiled at 95°C for 5 minutes. Proteins were then separated on a 12.5% SDS-PAGE gel. EZ-Run Prestained Rec protein ladder was used as a molecular weight marker. Proteins were transferred under semi-dry conditions to nitrocellulose membrane (0.45 μm Protran), labelled with the appropriate primary antibodies: anti-HA (1:500, Sigma), anti-ALD (1:2000), anti-Mys/TGME49_215430 ([81 (link)], 1:2000), anti-TOM40 ([48 (link)], 1:2000), anti-ATPβ (Agrisera AS05085, 1:5000), IMC1 ([82 (link)], 1:1000), CPN60 ([83 (link)], 1:2000), SAG1 (gift from the Soldati lab, 1:2000), and detected using either secondary horseradish peroxidase-conjugated antibodies and chemiluminescence detection using Pierce ECL Western Blotting Substrate and an x-ray film or using secondary fluorescent antibodies IRDye 800CW, 680RD (1:10000, LIC-COR) and detection with an Odyssey CLx.
For the blue native-PAGE, proteins were transferred onto a PVDF membrane (0.45 μm, Hybond) using wet transfer in Towbin buffer (0.025 M TRIS 0.192 M Glycine 10% Methanol) for 60 minutes at 100 V. After transfer, immunolabelling and visualisation was carried out as described above by chemiluminescence detection.
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3

Quantitative Western Blot Analysis of Protein Expression

The primary antibodies used were H+ ATPase (1:1,000; AS07 260) and AtpB (1:10,000; AS05 085) from Agrisera. The secondary antibody used was HRP-conjugated goat anti-rabbit IgG (1:100,000 for quantification of the condition-dependent PMA expression and 1:1500 for detection of the exogenous PMA protein; AS09 602) from Agrisera. The dilution rate of the secondary antibody was adjusted according to the manufacturer protocol of the developing reagents. In quantitative analysis of protein expression levels, the band intensity was measured using ImageJ software. Detailed experimental procedures are supplied in Supplementary Method 4.
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The samples for Western blot analysis were first subjected to SDS-PAGE using 12% acrylamide gel with 6M urea to separate proteins according to size. The samples were loaded based on the Bradford assay to 1–4μg protein per well (see each experiment for details), with 1:1 vol buffer containing 138mM Tris–HCl pH 6.8, 6M Urea, 22.2% glycerol, 4.3% SDS, 10% β-mercaptoethanol, and a small volume of Bromophenol blue. The separated proteins were electro-transferred from the acrylamide gel to Immobilon PVDF membrane (Millipore). The PVDF membrane was blocked with 5% milk (BIO-RAD blotting grade blocker) and incubated with first antibody and second antibody and finally with ECL Western blotting Detection Reagent (Amersham GE Healthcare) before imaging on film. The protein-specific antibodies used in the assay were α-ADO (custom made, see Generating ADO-specific antibody above) at 1:1000 dilution, α-PsaB (AS10 695, Agrisera) at 1:1000 dilution, and α-ATPase β (AS05 085, Agrisera) at 1:5000 dilution. The secondary antibody (AS09 602, HRP conjugated Goat anti-Rabbit IgG (H&L), Agrisera) was used at 1:10000 dilution.
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Proteins fractionated by gel electrophoresis were transferred to polyvinylidene difluoride membranes (PVDF; Immobilon-P, Millipore) using a semi-dry blotting system (BioRad) as described in the supplier’s instructions. After blocking with TBST (10 mM Tris/HCl pH 8.0, 150 mM NaCl and 0.1% [v/v] Tween-20) supplemented with 3% (w/v) skim milk powder, the membranes were incubated with antibodies at 4°C overnight. Antibodies used in this study were obtained from Agrisera (CF1-β: AS05 085, 1:5,000; CF1-γ: AS08 312, 1:5,000; CFO-b: AS10 1604, 1:5,000; CFO-c: AS09 591, 1:3,000; AtCGL160: AS12 1853, 1:1,000; PsbO: AS05 092, 1:10,000 and PsaD: AS09 461, 1:1,000).
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Immunoblot analysis was performed as described previously14 (link). Antibodies against ATPB (AS05 085, rabbit polyclonal) and firefly luciferase (M095-3, mouse monoclonal) were obtained from Agrisera (Sweden) and MBL (Japan), respectively. Rabbit polyclonal antibodies against LHCSR1 and both the LHCSRs were raised and affinity-purified against the peptide SGKRTVSGKAGAPVP or LGLKPTDPEELK, respectively (Eurofins Genomics, Japan).
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BS strands were isolated following the procedure of Ghannoum et al. (2005 (link)). Total protein extracts were isolated from 0.7 cm2 frozen leaf discs or from BS strands as described in Ermakova, Bellasio, et al. (2021 (link)), loaded on leaf area or protein basis and separated by SDS‐PAGE as described in Ermakova et al. (2019 (link)). Proteins were then transferred to a nitrocellulose membrane and probed with antibodies against various photosynthetic proteins in dilutions recommended by the producer: Rieske (AS08 330; Agrisera, Vännäs, Sweden), D1 (AS10 704; Agrisera), AtpB (AS05 085; Agrisera), PsbS (AS09 533; Agrisera), Lhcb2 (AS01 003; Agrisera), RbcL (Martin‐Avila et al., 2020 (link)), PEPC (Ermakova, Arrivault, et al., 2021 (link)). Quantification of immunoblots was performed with Image Lab software (Biorad, Hercules, CA).
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Parasite samples were resuspended in 1 × NuPAGE LDS loading dye (Invitrogen, Paisley, UK) supplemented with 5% v/v beta-mercaptoethanol or Laemmli buffer (2% (w/v) SDS, 125 mm Tris–HCl pH 6.8, 10% (w/v) glycerol, 0.04% (v/v) β-mercaptoethanol, and 0.002% (w/v) bromophenol blue), heated at 95°C for 5 min and were separated on a SDS-PAGE gel, using a EZ-Run Prestained Rec protein ladder as a molecular weight marker. Proteins were transferred under semi-dry conditions in Towbin buffer (0.025 M Tris 0.192 M Glycine 10% Methanol) onto nitrocellulose membrane (0.45 μm Protran, Merck, Gillingham, UK). Membranes were then labelled with the relevant primary antibodies: anti-HA (1:500, rat, Sigma, Merck, Gillingham, UK), anti-TOM40 (1:2000, rabbit, [30 (link)]), anti-Ty (1:1000, mouse, [57 (link)]) and anti-ATPβ (1:2000, rabbit, Agrisera AS05 085) before being labelled with secondary fluorescent antibodies IRDye 800CW and IRDye 680RD (1:10,000, LIC-COR, Lincoln, NE, USA) and visualized with Odyssey CLx imaging system.
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9

Protoplast Transformation and Chloroplast Protein Extraction

Fifty micrograms of OsPPR676 and 50 μg of Osj10gBTF3 or OsHSP82 plasmids were co-transfected into rice protoplasts (1 × 105), which were isolated from WT, hsp82-1, or btf3-1 mutant seedlings at 12 days according to Guidelines for biological experiments in rice. The transformation was performed by 40% PEG and incubated in WI solution for 5 h. Samples were collected before 10 μm CHX was added, and at 3 h after 10 μm CHX was added, FLAG antibody (DK3201, Elabscience).
Chloroplast proteins were extracted from leaf tissues of 2-week-old WT, hsp82-1, and btf3-1 seedlings as described previously (Cho et al., 2006 (link)). Coomassie Blue staining was used as an internal loading control. The target protein, atpB, was detected by atpB antibody (AS05085, Agrisera). The nuclear protein histone and cytoplasmic protein GAPDH were detected by histone (D151717, Sangon Biotech) and GAPDH (D110016, Sangon Biotech) antibody, respectively.
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Mitochondrial protein extraction and Western blotting were done as previously reported (Yang et al., 2022a (link)). The abundance of ATP6 and ATPβ proteins was detected using anti‐ATP6 (Biogot technology, China)/anti‐ATPβ (AS05085; Agrisera, Uppsala, Sweden) antiserum diluted to 1:1000. COXII (1:2000, AS04053A; Agrisera, Uppsala, Sweden) was used as protein loading control. Anti‐ATP6 antibody can recognize both ATP6 and ATP6C proteins because the immunogen sequence used in antibody generation is in a shared region of the two proteins. Other mitochondrial antibodies we used included anti‐Nad7 (1:2000, PHY05138S; PHYTOAB, California, USA), anti‐SDH1 (1:2000, PHY0558S; PHYTOAB, California, USA), anti‐CYC1 (1:2000, PHY0566S; PHYTOAB, California, USA).
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