The EpiQuik Plant ChIP Kit (Epigentek) was used for ChIP assays. Briefly, 10-day-old OsMADS23-GFP seedlings were harvested and fixed in 1% formaldehyde, and chromatin was isolated from 2 g crosslinked leaves. Isolated chromatin was sonicated for DNA fragmentation ranging from 200 to 500 bp. Subsequently, the DNA/protein complex was immunoprecipitated with anti-GFP antibody (Abmart, M20004) or IgG. Then the immunoprecipitated DNA was purified with phenol/chloroform after reverse crosslinking and proteinase K treatment. The immunoprecipitated DNA was used for qPCR analysis. The primers for ChIP-qPCR used are listed in S1 Table .
M20004
Manufactured by Abmart
Sourced in China
The M20004 is a laboratory centrifuge designed for general purpose applications. It features a maximum speed of 6,000 rpm and an adjustable timer. The centrifuge accommodates a variety of sample sizes and volumes. Further details on its specific functionality and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
M20008, by Abmart (3 mentions)
M20003, by Abmart (2 mentions)
M20002, by Abmart (2 mentions)
Phosstop, by Roche (2 mentions)
Epiquik plant chip kit, by Epigentek (1 mentions)
Streptavidin hrp, by Cell Signaling Technology (1 mentions)
Anti myc antibody, by Cell Signaling Technology (1 mentions)
Anti ha antibody, by Cell Signaling Technology (1 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (1 mentions)
Anti pmp70, by Merck Group (1 mentions)
Ab27671, by Abcam (1 mentions)
Sab4200181, by Merck Group (1 mentions)
Anti myc hrp, by Cell Signaling Technology (1 mentions)
Anti ha hrp, by Cell Signaling Technology (1 mentions)
Anti gfp hrp, by Cell Signaling Technology (1 mentions)
Anti β actin, by GenScript (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
14 protocols using m20004
1
ChIP Assay for OsMADS23-GFP Seedlings
2
Immunoblotting Antibody Detection Protocol
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Detecting reagents, including streptavidin-HRP (1:2,000 for WB, 3999S), anti-Myc antibody (1:2,000 for WB, 2276S), anti-Myc HRP (1:1,000 for WB, 2040S), anti-HA antibody (1:2,000 for WB, 2367S), anti-HA HRP (1:1,000 for WB, 2999S), V5 (1:1,000 for immunofluorescence, 13202S), and anti-GFP HRP (1:1,000 for WB, 2037S) were purchased from Cell Signaling Technology. Antibodies against V5 (1:3,000 for WB, ab27671) and MARCH5 (1:2,000 for WB, ab174959) were purchased from Abcam. Anti-GDAPH (1:4,000 for WB, A00191) and anti–β-actin (1:4,000 for WB, A00702) were purchased from GenScript. Other antibodies used in this study included anti-FLAG (1:2,000 for WB, GNI14110-FG) and anti-FLAG HRP (GNI4310-FG; GNI), anti-PMP70 (1:3,000 for WB, SAB4200181; Sigma-Aldrich), and anti-GFP (1:3,000 for WB, M20004; Abmart). Anti–V5-HRP (1:5,000 for WB, R961-25) was purchased from Thermo Fisher Scientific. Other primary antibodies used were anti-MARCH5 (19168S; Cell Signaling Technology), anti-PEX19 (14713–1-AP; Proteintech), anti-PEX3 (sc-271477; Santa Cruz Biotechnology), anti-catalase (219010; Millipore), and anti-PEX13 (ab235043; Abcam). Secondary antibodies conjugated to HRP were purchased from GenScript.
3
Immunoblotting and Immunofluorescence Assays
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The primary antibodies used are: rabbit anti-SCAMP5 (ab3432, Abcam;1:500 for WB); rabbit anti-LC3B (L7543, Sigma-Aldrich; 1:3000 for WB); mouse anti-p62/SQSTM1 (ab56416, Abcam; 1:2000 for WB); rabbit anti-TFEB (13372-1-AP, Proteintech; 1:1000 for WB); mouse anti-α-synuclein (610787, BD Biosciences; 1:1000 for WB); rabbit anti-huntingtin (5656P, Cell signaling; 1:1000 for WB); mouse anti-GM130 (610822, BD Biosciences; 1:300 for IF); mouse anti-Giantin (ab37266, Abcam; 1:300 for IF); rabbit anti-ERGIC53 (sc-66880, Santa Cruz; 1:100 for IF); rabbit anti-Calnexin (ab22595, Abcam; 1:2000 for WB); mouse anti-CD63 (ab8219, Abcam; 1:1000 for WB); mouse anti-DYKDDDDK-Tag (FLAG) (M20008, Abmart; 1:3000 for WB; 1:300 for IF; 1:200 for IP); goat anti-DYKDDDDK-Tag (FLAG) (NB600-344, NOVUS; 1:100 for IF); mouse anti-HA-Tag (M20003, Abmart; 1:200 for IP); mouse anti-GFP-Tag (M20004, Abmart; 1:2000 for WB); mouse anti-β-tubulin (M20005, Abmart; 1:3000 for WB); mouse anti-β-actin (M30002, Abmart; 1:2000 for WB).
The drugs used are: Rapamycin (553210, Merck, 100nM); Bafilomycin A1 (196000, Merck, 10-50nM); MG132 (474790, Merck, 3-5μM); Cycloheximide (C7698, sigma, 100μg/ml); Tetanus toxic (T3194. Sigma-Aldrich, 2nM); BrefeldinA (S1536, Beyotime, 2μM).
The drugs used are: Rapamycin (553210, Merck, 100nM); Bafilomycin A1 (196000, Merck, 10-50nM); MG132 (474790, Merck, 3-5μM); Cycloheximide (C7698, sigma, 100μg/ml); Tetanus toxic (T3194. Sigma-Aldrich, 2nM); BrefeldinA (S1536, Beyotime, 2μM).
4
Western Blot Analysis of Protein Targets
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The samples (cells, NVs, and animal
tissues) were lysed in RIPA lysate buffer. The protein samples were
placed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) gel and separated in the electrophoresis tank. The
protein signal was then transferred from the SDS–PAGE gel to
the polyvinylidene (PVDF) film which was later soaked in skimmed milk
powder and sealed at room temperature. After rinsing with TBST buffer,
the primary antibody and the corresponding resistant secondary antibody
were added for incubation. Finally, ECL was covered on the PVDF film
for exposure. Primary antibodies used were GFP (Abmart, M20004), TNF-R1
(Abmart, TA0282), p105/p50 (Abmart, T55040), and β-actin (Abmart,
T40104).
tissues) were lysed in RIPA lysate buffer. The protein samples were
placed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) gel and separated in the electrophoresis tank. The
protein signal was then transferred from the SDS–PAGE gel to
the polyvinylidene (PVDF) film which was later soaked in skimmed milk
powder and sealed at room temperature. After rinsing with TBST buffer,
the primary antibody and the corresponding resistant secondary antibody
were added for incubation. Finally, ECL was covered on the PVDF film
for exposure. Primary antibodies used were GFP (Abmart, M20004), TNF-R1
(Abmart, TA0282), p105/p50 (Abmart, T55040), and β-actin (Abmart,
T40104).
5
Co-Immunoprecipitation of DGS1 and SMG3
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The coding sequences of DGS1 was fused to the Kpn I and BamH I sites of pCambial1300-221-Myc to generate Pro35S:Myc-DGS1. Pro35S:GFP-SMG3 and Pro35S:Myc-DGS1 were cotransformed into N. benthamiana leaves for transient expression. Total protein of the transformed leaves was extracted using a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 2% TritonX-100, 20% glycerol, 1 mM EDTA, and 1 tablet protease inhibitor cocktail (Roche) per 50 ml buffer. Then, the Co-IP assay was performed as previously described (Hao et al., 2021) . Briefly, the extracted total protein was incubated with GFP-beads (GFP-Trap® Agarose, Chromo Tek) for 1 h at 4°C, and then the GFP-beads were washed 5 times with wash buffer (50 mM Tris-HCl, 150 mM NaCl, 0.01% TritonX-100, 20% glycerol, 1 mM EDTA, and 1 tablet protease inhibitor cocktail (Roche) per 50 ml buffer). Then, beads were denatured in protein loading buffer (98°C, 5 min) and subjected to SDS-PAGE. Anti-GFP (Abmart; Catalog, M20004; Dilution 1:5000) and anti-MYC (Abmart; Catalog, M20002; Dilution 1:5000) were used to detect the input and immunoprecipitated proteins.
6
Co-IP Assay for Transcription Factor Interactions
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For Co-IP assays, the 35S:SlWRKY30-GFP, 35S:SlWRKY52-HA, 35S:SlWRKY59-HA, 35S:SlWRKY80-HA, 35S:SlWRKY81-HA, and 35S:HA (as a negative control) constructs were transformed into A. tumefaciens (GV3101). N. benthamiana leaves were infiltrated with A. tumefaciens suspensions containing the respective constructs, followed by incubation at 25°C for 36–48 hours (16-hours light/8-hours dark cycle). Subsequently, total proteins were extracted from the samples using protein extraction buffer [50 mM Tris–HCl (pH 7.5), 150 mM MaCl, 10 mM MgCl2, 0.1% (v/v) Tween 20, 1 mM PMSF, and 1 × protease inhibitor cocktail (Roche)] and incubated with magnetic GPF-trap beads (Chromotek, Germany) at 4°C for 2 hours. After washing three times with Co-IP buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 0.25% IGEPAL CA-630, and 1 mM PMSF], the samples were separated through 12% SDS–PAGE. For immunoblot analysis, anti-GFP (M20004, Abmart, Shanghai, China, 1/5000) and anti-HA (M20003, Abmart, Shanghai, China, 1/3000) antibodies were used to detect the levels of SlWRKY30 or SlWRKY52/59/80/81proteins, respectively.
7
Western Blotting and Immunofluorescence Assays
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Mouse anti-CFAP52 polyclonal antibody (aa 421–620) was generated by Quan Biotech and was used at a 1:500 dilution for Western blotting. Rabbit anti-CFAP45 antibody (HPA043618, Atlas Antibodies) was used at a 1:500 dilution for Western blotting and a 1:400 dilution for immunofluorescence. Mouse anti-GAPDH antibody (AC002, ABclonal) was used at a 1:5000 dilution for Western blotting. Rabbit anti-Tubulin antibody (A6830, ABclonal) was used at a 1:5000 dilution for Western blotting. Mouse anti-GFP antibody (M20004, Abmart) was used at a 1:2000 dilution for Western blotting. Rabbit anti-MYC antibody (BE2011, EASYBIO) was used at a 1:2000 dilution for Western blotting. Rabbit anti-AKAP4 antibody (A14813, ABclonal) was used at a 1:100 dilution for immunofluorescence. Mouse anti-acetylated α tubulin antibody (T7451, Sigma-Aldrich) was used at a 1:200 dilution for immunofluorescence. The secondary antibodies were goat anti-rabbit FITC (1:200, ZF-0311, Zhong Shan Jin Qiao), goat anti-mouse FITC (1:200, ZF-0312, Zhong Shan Jin Qiao), and goat anti-rabbit TRITC (1:200, ZF0313, Zhong Shan Jin Qiao). MitoTracker Deep Red 633 (1:1500 dilution, M22426, Thermo Fisher Scientific) was used for immunofluorescence.
8
Western Blot Analysis of GFP and FLAG Proteins
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Agroinfiltrated N. benthamiana leaves were harvested at 48 hpi, frozen in liquid nitrogen, ground to a fine powder, and mixed with the extraction buffer (50 mM HEPES, 150 mM KCl, 1 mM EDTA, 0.1% Triton X‐100, pH 7.5) supplemented with 1 mM dithiothreitol (DTT) and protease inhibitor cocktail (Sigma‐Aldrich). The proteins were fractionated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to 0.2‐μm polyvinylidene difluoride (PVDF) membranes (Millipore Sigma Co. Ltd) presoaked in methanol for 15 s. Nonspecific protein binding was blocked by shaking the membranes at 40 rpm with Tris‐buffered saline (TBS; pH 7.4) containing 3% non‐fat dry milk for 1 h at 25°C. Anti‐GFP (1:5000 dilution; #M20004) and anti‐FLAG (1:5000 dilution; #M20008, both from Abmart Inc.), or anti‐RFP (1:1000 dilution; #5f8, ChromoTek) antibodies were added to the blocking buffer and incubated at room temperature for 60–90 min. After three washes with TBS containing 0.1% Tween 20 (TBST) for 5 min each, the membranes were incubated with goat antimouse antibody (1:10,000 dilution; Odyssey no. 926‐32210; Li‐Cor Biosciences) in TBST at room temperature for 45 min. The membrane was washed three times with TBST and blotted.
9
Co-immunoprecipitation Assay for Protein Interactions
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WT and PC1 overexpressing rice seedlings were used for Co-IP assays. For Co-IP assays in N. benthamiana, the constructs Ubipro:CatC-FLAG, 35S:PC1-GFP, and 35S:GFP were transiently expressed in 3-wk-old N. benthamiana leaves by Agrobacterium-mediated infiltration. The Co-IP assay was performed as described previously (Feng et al. 2008) (link). Anti-GFP beads (Sigma-Aldrich, USA) were used to immunoprecipitate protein complexes. The immunoprecipitated proteins were subsequently released by boiling in 2.5× SDS sample buffer and subjected to 10% SDS-PAGE. The anti-GFP (M20008, Abmart, China, dilutions: 1:5,000) and anti-CAT antibodies were used for immunoblot analysis of the samples derived from rice. The anti-GFP and anti-FLAG antibodies (M20004, Abmart, China, dilutions: 1:5,000) were used for immunoblot analysis of the samples derived from N. benthamiana.
10
Chromatin Extraction and Immunoprecipitation of OsNAC120-GFP
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Chromatin extraction and immunoprecipitation were performed as described previously (Saleh et al., 2008 (link)) with minor modifications. In brief, chromatin was isolated from crosslinked leaves of 2-week-old OsNAC120-GFP transgenic plants. Isolated chromatin was sonicated to obtain DNA fragments ranging from 200 to 500 bp. DNA–protein complexes were immunoprecipitated with anti-GFP antibody (1:2500, Abmart, M20004), and the immunoprecipitated DNA fragments were detected by qPCR analysis with gene-specific primers. Primers used are listed in supplemental Table 1 .
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