Western blots were performed as described63 (link). Proteins from 14-day-old seedlings were extracted, resolved in 12% (v/v) SDS-polyacrylamide gels, and transferred to Hybond C-Extra membranes (Amersham Biosciences). The membranes were blocked with 5% (w/v) non-fat milk in Tris buffered saline tween (TBST) buffer and then probed with specific antibodies. Antibodies used included anti-GAPDH (Santa Cruz Biotechnology, sc-365062, dilution, 1:1000), anti-SUL (dilution, 1:1000)35 (link), anti-HYL1 (Agrisera, AS06136, dilution, 1:2000), anti-AGO1 (Agrisera, AS09527, dilution, 1:2000), anti-SE (Agrisera, AS09532A, dilution, 1:1000), anti-H3 (Agrisera, AS10710, dilution 1:3000) and anti-GFP (Abcam, ab290, dilution, 1:3000). After three washes, the membranes were probed with horseradish peroxidase-conjugated, goat-anti-rabbit IgG (Bio-Rad, cat.172-1019) (dilution, 1:2000) or goat anti-mouse IgG (Bio-Rad, cat.170-6516, dilution, 1:2000). The protein signals were detected with the AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE healthcare, RPN2232) and visualized with the Chemiluminescence imaging system (Clinx Science Instruments Co. Ltd., China).
AS09532A
Manufactured by Agrisera
Sourced in Sweden
AS09532A is a lab equipment product offered by Agrisera. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
AS06136, Agrisera (6 mentions)
AS122102, Agrisera (3 mentions)
AS09527, Agrisera (2 mentions)
A4596, Merck Group (2 mentions)
A8592, Merck Group (2 mentions)
ADI-SPA-818, Enzo Life Sciences (2 mentions)
Sc-2005, Santa Cruz Biotechnology (2 mentions)
AS14 2785, Agrisera (2 mentions)
AS19 4307, Agrisera (2 mentions)
AS09 531, Agrisera (2 mentions)
AS09 602, Agrisera (2 mentions)
Ab290, Abcam (1 mentions)
Hybond-C Extra membranes, GE Healthcare (1 mentions)
AmershamTM ECLTM Prime Western Blotting Detection Reagent, GE Healthcare (1 mentions)
Sc-365062, Agrisera (1 mentions)
Goat anti-mouse IgG, Bio-Rad (1 mentions)
Goat anti-rabbit IgG, Bio-Rad (1 mentions)
Gta-20, Proteintech (1 mentions)
Ab190584, Abcam (1 mentions)
M4439, Merck Group (1 mentions)
11 protocols using AS09532A
1
Western Blot Analysis of Plant Proteins
2
ChIP-qPCR Analysis of MdNCED3 Promoter
ChIP-qPCR assays were performed as described previously13 . Tissue-cultured GL-3 was used for crosslinking, and the ChIP assay was performed with an anti-SE antibody (Agrisera, AS09 532A). Three regions of the MdNCED3 promoter were examined by qPCR, with no antibody ChIP samples serving as the reference. The primers used for ChIP-qPCR are listed in Supplementary Table 1 .
3
Investigating JANUS Interactions with DCL1 and SE
To examine the interaction of JANUS with DCL1 and SE, JANUS-FLAG was transiently co-expressed with GFP-DCL1 or MYC-SE in N. benthamiana as described (12 (link)). The expression of these transgenes was directed by the 35S promoter. Total proteins of infiltrated leaves were extracted with an extraction buffer (50 mM Tris–HCl 8.0, 150 mM NaCl, 5% glycerol, 5% Triton X-100, 1 mM EDTA, 1× complete protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitation (IP) was performed on protein extracts using anti-GFP (GTA-20, Chromotek) or anti-FLAG antibodies (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were separated on a 10% SDS-PAGE and detected with western blot using monoclonal antibodies against GFP (902602, Biolegend) or FLAG (A8592, Sigma). DCL1, SE and HYL1 proteins in Arabidopsis were detected with antibodies against DCL1 (Agrisera, AS122102), SE (Agrisera, AS09532A) and HYL1 (Agrisera, AS06136).
4
Co-Immunoprecipitation Assay for Protein Interactions
The Co-IP assay was performed as previously described66 (link). To examine the interactions of CWC15 with HYL1, SE, PAB1 and PAG1, MYC-CWC15 was transiently co-expressed with HYL1-2HA, SE-2HA, PAB1-2HA and PAG1-2HA in N. benthamiana leaves, respectively. The interaction between CWC15 and PRP4KA was performed by co-expressing 2HA-CWC15 and FLAG-PRP4KA in N. benthamiana. IP was performed on protein extracts using anti-HA (26181, Thermo Scientific), anti-MYC (GTA020, Bulldog Bio) or anti-FLAG (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were detected with Western blot using antibodies against MYC (1:5000 dilutions, rabbit monoclonal, M4439, Sigma), FLAG (1:10000 dilutions, mouse monoclonal, A8592, Sigma) or HA (1:5000 dilutions, mouse monoclonal, H6533, Sigma). For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant. After IP, proteins were detected with Western blot using anti-GFP (1:1000 dilutions, rabbit polyclonal, ab190584, Abcam), anti-HYL1 (1:5000 dilutions, rabbit polyclonal, AS06136, Agrisera) or anti-SE (1:5000 dilutions, rabbit polyclonal, AS09532A, Agrisera) antibodies.
5
Arabidopsis Inflorescence Protein Analysis
6
Western Blot Analysis of Plant and Yeast Proteins
Western blot analysis was typically performed with 10-day-old plants that were grown on an MS plate or 3-week-old plants that were grown on soil. The extraction and experimental procedures were performed as described (59 (link)). The primary antibodies were used against Myc (Sigma-Aldrich, C 3956), HA (Sigma-Aldrich, H9658), actin (Sigma-Aldrich, A0480), His (Sigma-Aldrich, H1029), SE (Agrisera, AS09 532A), DCL1 (Agrisera, AS12 2102), AGO1 (Agrisera, AS09 527), and HYL1 [from S. W. Yang’s laboratory (53 (link))]. Secondary antibodies used were goat-developed anti-mouse immunoglobulin G (GE Healthcare, NA931) and anti-rabbit (GE Healthcare, NA934).
For Western blot analysis of yeast extracts, an amount of 105 yeast cells was harvested, resuspended in 200 μl of lysis buffer [0.1 M NaOH, 0.05 M EDTA (pH 8.0), 2% SDS, and 2% β-mercaptoethanol], and boiled in 95°C for 10 min. The cell resuspension was supplied with 5 μl of 4 M acetic acid into and boiled in 95°C for 10 min. Then, the cell resuspension was mixed with 50 μl of loading buffer [0.25 M tris-HCl (pH 6.8), 50% glycerol, and 0.05% bromophenolblue).
For Western blot analysis of yeast extracts, an amount of 105 yeast cells was harvested, resuspended in 200 μl of lysis buffer [0.1 M NaOH, 0.05 M EDTA (pH 8.0), 2% SDS, and 2% β-mercaptoethanol], and boiled in 95°C for 10 min. The cell resuspension was supplied with 5 μl of 4 M acetic acid into and boiled in 95°C for 10 min. Then, the cell resuspension was mixed with 50 μl of loading buffer [0.25 M tris-HCl (pH 6.8), 50% glycerol, and 0.05% bromophenolblue).
7
Arabidopsis Leaf Protein Analysis
Thirty micrograms of Arabidopsis whole leaf extract (extraction buffer: 100 mM Tris–HCl pH 7.5, 10% glycerol, 5 mM EDTA, 5 mM EGTA, 0.15 M NaCl, 0.75% Triton X-100, 0.05% SDS, and 1 mM DTT) was resolved in a 10% denaturing gel, transferred to PVDF membranes, and detected by immunoblotting. The following antibodies were used: anti-AtPRP40b (AS14 2785, Agrisera; 1:10,000), anti-DCL1 (AS19 4307; Agrisera; 1:100), anti-CBP80 (AS09 531; Agrisera: 1:2,000), anti-SE (AS09 532A; Agrisera; 1:2,000), anti-HYL1 (AS06 136; Agrisera: 1:1,000), anti-actin (691001, MP Biomedicals; 1:1,000), anti-rabbit (AS09 602, Agrisera; 1:20,000), and anti-mouse (sc-2005, Santa Cruz Biotechnology, 1:10,000).
8
Immunoblot Analysis of Cellular Fractions
Immunoblot analysis of total lysate, cytoplasmic and nucleoplasmic fractions was essentially performed as described [29 (link)]. Briefly, primary antibodies were polyclonal antibodies against HISTONE 3 (Agrisera, Vännäs, Sweden, AS10710; rabbit; dilution 1:5000), PHOSPHOENOLPYRUVATE CARBOXYLASE (Agrisera AS09458; rabbit; dilution 1:20,000) and SERRATE (Agrisera AS09532A; rabbit; dilution 1:1000). Secondary antibody was HRP-coupled anti-rabbit IgG (Sigma-Aldrich, Burlington, MA, USA, A0545; dilution 1:2500). Chemiluminescence detection of the immunoblots was done with Fusion-FX6 (Vilber) system.
9
Protein Extraction and Western Blot Analysis
Plant samples were ground in liquid nitrogen, and total proteins were isolated by extraction buffer [0.2 M NaCl, 5 mM MgCl2, 5 mM DTT, 20 mM tris-HCl (pH 7.5), 0.03% Tween 20 (Ameresco), and 0.5 tablets of protease inhibitor (Roche)]. The supernatant was collected by centrifuging at 12,000 rpm for 15 min. Total proteins were examined by Western blot analysis using α-tubulin (1:5000; EASYBIO, BE0031) as a loading control.
Proteins in the study were also probed with α-RH12 (1:2000; GenScript, GTGRGAPPNPDYHQC), α-SE (1:2000; Agrisera, AS09532A), α-HYL1 (1:2000; Agrisera, AS06136), α-GFP (1:2000; EASYBIO, BE2001), α-FLAG (1:2000; Sigma-Aldrich, F7425), α-HA (1:2000; EASYBIO, BE7002), α-TuMV-VPg (1:200; GenScript, KGKSKGRTRGIGHKC), α-TuMV-CP (1:5000; GenScript, AGETLDAGLTDEQKC), α-TuMV-HC-Pro (1:2000), α-CMV-CP (1:3000), and α-MEMB12 (1:2000) (23 (link)). Secondary antibodies were goat anti-rabbit IgG (1:5000; EASYBIO, BE0101) and goat anti-mouse IgG (1:5000; EASYBIO, BE0102).
Proteins in the study were also probed with α-RH12 (1:2000; GenScript, GTGRGAPPNPDYHQC), α-SE (1:2000; Agrisera, AS09532A), α-HYL1 (1:2000; Agrisera, AS06136), α-GFP (1:2000; EASYBIO, BE2001), α-FLAG (1:2000; Sigma-Aldrich, F7425), α-HA (1:2000; EASYBIO, BE7002), α-TuMV-VPg (1:200; GenScript, KGKSKGRTRGIGHKC), α-TuMV-CP (1:5000; GenScript, AGETLDAGLTDEQKC), α-TuMV-HC-Pro (1:2000), α-CMV-CP (1:3000), and α-MEMB12 (1:2000) (23 (link)). Secondary antibodies were goat anti-rabbit IgG (1:5000; EASYBIO, BE0101) and goat anti-mouse IgG (1:5000; EASYBIO, BE0102).
10
Arabidopsis Inflorescence Protein Analysis
Protein extracts from inflorescences of Arabidopsis were separated on a 10% Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected with antibodies against HYL1 (Agrisera, AS06136), SE (Agrisera, AS09532A), DCL1 (Agrisera, AS122102), AGO1 (Agrisera, AS09527) and HSC70 (Enzo, ADI-SPA-818).
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