Cell extracts were prepared by using the following lysis buffer (4% sodium dodecyl sulfate, 20% glycerol, 100 mM dithiothreitol, Tris–HCl, pH 6.8). In total, 20 μg of proteins were loaded and separated by 10 or 15% sodium dodecyl sulfate-polyacryl-amide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane (Bio-Rad, Richmond, CA, USA). Then, 5% non-fat milk in Tris-buffered saline was used to block the membrane and immunoblotted with antibodies against Flag (1:1,000, Sigma-Aldrich, A8592), Actin (1:10,000, Merck, MAB1501), HIF-1α (1:1,000, BD Transduction Laboratories, 610958), VHL (1:500, Novus biologicals, NB100-485), ALDH2 (1:1,000, Abgent, AM1831a), PDK1 (1:1,000, Stressgen, ADI-KAP-PK112-D) and HNF-4α (1:500, Santa Cruz, sc-6556). Followed by horseradish peroxidase-linked second antibody (1:2,000, Cell signaling Technology, Beverly, MA, USA) for 1 h at room temperature, detection was performed by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. All the uncropped versions of images were shown in Supplementary Fig. 10 .
A8592
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
The A8592 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the A8592 is to provide a reliable and consistent platform for various laboratory procedures and experiments. Further details on the specific intended use or capabilities of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
H6533, by Merck Group (4 mentions)
Ab85961, by Abcam (3 mentions)
Ab1791, by Abcam (3 mentions)
A5441, by Merck Group (3 mentions)
A4596, by Merck Group (3 mentions)
T9026, by Merck Group (3 mentions)
Optiprep cushion, by Merck Group (3 mentions)
G8795, by Kerafast (3 mentions)
Bis tris page gel, by Thermo Fisher Scientific (3 mentions)
A0545, by Merck Group (2 mentions)
Anti flag m2 hrp, by Merck Group (2 mentions)
F3165, by Merck Group (2 mentions)
Anti flag hrp, by Merck Group (2 mentions)
M4439, by Merck Group (2 mentions)
Anti ha affinity resin, by Merck Group (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
As06136, by Agrisera (2 mentions)
As09532a, by Agrisera (2 mentions)
V2260, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
73 protocols using a8592
1
Western Blot Analysis of Protein Expression
2
MAPK Phosphorylation Assay
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Two-week-old seedlings grown in liquid MS media (MAPK phosphorylation) or leaf disks from 4-week-old plants were flash-frozen in liquid nitrogen. Frozen plant tissue was ground in a Genogrinder with 2 mm glass beads (1500 strokes/min, 1.5 min) prior to boiling in 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris-HCl; 10 μL/mg tissue) for 10 min at 95°C. The samples were then spun at 13,000 rcf for 5 min prior to loading and running on SDS-PAGE gels. Proteins were transferred using semi-dry transfer onto PVDF membrane (ThermoFisher), blocked in 5% (w/v) bovine serum albumin prior to incubation with appropriate antibodies α-pMAPK (p44/42 MAPK [Erk1/2] antibody #9102; 1:4000); α-FLAG-HRP (A8592, Merck; 1:5000); and α-rabbit-HRP (A-0545, Merck; 1:10,000). Western blots were imaged with a LAS 4000 IMAGEQUANT SYSTEM (GE Healthcare) or on X-ray film before being developed. Staining of the blotted membrane with Coomassie brilliant blue was used to confirm loading.
3
Overexpression of LSD1 in Zebrafish
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Capped mRNAs (500 pg) transcribed from linearized pCS2 plasmids were injected into one-cell-stage embryos as previously described13 (link). Overexpressed LSD1 proteins at 15 hpf were detected by immunoblotting using anti-FLAG antibody attached with horseradish peroxidase (A8592, Merck) as previously described30 (link). Total protein loading was confirmed by Coomassie brilliant blue (CBB) staining.
4
Western Blot Analysis of Transcription Factors
5
Chromatin Regulation Protocols in Cell Lines
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Eco-Pack 2–293 cells (Clontech) and 293T cells (gifted from Dr. Yao [Ito et al., 2001 (link)]) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum. Wild-type and Suv39h dn iMEFs (Lachner et al., 2001 (link)) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 0.1 mM beta-mercaptoethanol, and 1x non-essential amino acids. The following antibodies were used in this study: anti-FLAG-M2 (F3165: Sigma-Aldrich, RRID:AB_259529 ), anti-FLAG-M2-HRP (A8592: Sigma-Aldrich, RRID: AB_439702 ), anti-α-tubulin (T5168: Sigma, RRID: AB_477579 ), anti-Suv39h1 (8729: Cell Signaling, RRID: AB_10829612 ), anti-H3 (ab21054: Abcam, RRID: AB_880437 ), anti-H3K9me3 (ab8898: Abcam, RRID: AB_306848 and 2F3 (RRID: AB_2616099 )(Chandra et al., 2012 (link)), anti-HP1β (BMP002: MBL, RRID: AB_843158 ), and anti-GST (27-4577-01: GE Healthcare, RRID: AB_771432 ).
6
Protein Extraction and Western Blotting
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Cells were homogenized, and extracts were prepared using lysis buffer (NER buffer from the NER-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), 1× protease inhibitor cocktail, 1 μM microcystin-LR). The soluble fraction was recovered by centrifugation at 16,000 × g for 10 min at 4 °C. Protein concentration was measured with the BCA Protein Assay Kit (Pierce Biotechnology), and 30 μg of protein from each sample was resolved by SDS-PAGE. The resolved bands were transferred onto a nitrocellulose membrane and subjected to western blotting with the appropriate antibodies.
The following primary antibodies were used: mouse anti-CK1δ (C-8) (1/1000, Santa Cruz Biotechnology, sc-55553), goat anti-CK1δ (N-19) (1/500, sc-6475, Santa Cruz Biotechnology), rabbit-anti C-terminal Brd4 (1/2000)19 (link), rabbit anti-Brd4 (1/1000, A301-985A50, Bethyl Laboratories Inc.), rabbit anti-phospho-Brd4 492/494 (1/2000, ABE1453, Millipore), mouse anti-Gapdh (1/5000, NB300-221, Novus Biolechne), goat anti-CK2α (1/500, sc-6479, Santa Cruz Biotechnology), mouse anti-Flag (1/5000, A8592, Sigma), and rabbit anti-cyclin D1 (1/1000, ab134175, Abcam). The following secondary antibodies were used: anti-goat IgG–HRP (1/1000, 7074, Cell Signaling), anti-mouse IgG–HRP (1/1000, NXA931, GE Healthcare) and anti-rabbit IgG–HRP (1/1000, NA9340V, GE Healthcare).
The following primary antibodies were used: mouse anti-CK1δ (C-8) (1/1000, Santa Cruz Biotechnology, sc-55553), goat anti-CK1δ (N-19) (1/500, sc-6475, Santa Cruz Biotechnology), rabbit-anti C-terminal Brd4 (1/2000)19 (link), rabbit anti-Brd4 (1/1000, A301-985A50, Bethyl Laboratories Inc.), rabbit anti-phospho-Brd4 492/494 (1/2000, ABE1453, Millipore), mouse anti-Gapdh (1/5000, NB300-221, Novus Biolechne), goat anti-CK2α (1/500, sc-6479, Santa Cruz Biotechnology), mouse anti-Flag (1/5000, A8592, Sigma), and rabbit anti-cyclin D1 (1/1000, ab134175, Abcam). The following secondary antibodies were used: anti-goat IgG–HRP (1/1000, 7074, Cell Signaling), anti-mouse IgG–HRP (1/1000, NXA931, GE Healthcare) and anti-rabbit IgG–HRP (1/1000, NA9340V, GE Healthcare).
7
In vitro Ubiquitination Assay Protocol
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Ubiquitination assay was performed by following the method described previously with slight modification (Chen et al. 2021 (link)). Each 30 μL reaction mixture contained 50 ng recombinant wheat (Triticum aestivum) ubiquitin-activating enzyme E1 (GI: 136632), 100 ng human ubiquitin-binding enzyme E2 (UBCH5b), 5 µg ubiquitin protein (UBQ14, At4g02890), 500 ng GST-REIW1-3myc, and 2 μg OsWRKY31 substrate or its mutant in a reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM DTT, and 5 mM ATP) and incubated at 30 °C for 1 h. The reaction was stopped by adding 5× SDS loading buffer. Samples were separated by 10% SDS-PAGE gel and analyzed by immunobloting with anti-Ubiquitin (Sigma-Aldrich, U0508, 1:10,000 dilution) and HRP-conjugated anti-Flag (Sigma-Aldrich, A8592, 1: 10,000 dilution) antibodies.
8
Subcellular Fractionation and Western Blot Analysis
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In all, 3 × 106 cells were used for the extraction of nuclear and cytoplasmic proteins with the subcellular protein fractionation kit for cultured cells (78840, ThermoFisher Scientific) in accordance to the manufacturer’s instructions. In all, 10 µg of the nuclear and cytoplasmic fractions were then analysed by SDS-PAGE61 (link), and the following primary antibodies were used: WDR26 (ab85961, Abcam, 1:2000), NOLC1 (sc-374033, Santa Cruz, 1:1000), FLAG (A8592, Sigma, 1:1000), H3 (ab1791, Abcam, 1:2000), and HSP90 (ab13495, Abcam, 1:5000).
9
Western Blot Characterization of OCC-1 in CRC
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For detection of endogenous OCC-1 polypeptide in CRC cells, western blot was performed according to the previous report in which the polypeptide was identified (31 (link)) using three commercially available primary antibodies (ab83945, ab83948 and ab177759, Abcam) raised against three different regions of human OCC-1 polypeptide.
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
10
Ultrastructural and Protein Localization
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The experiments are performed as our previous described procedures. Thirty-nine EM images were obtained from thin sections using a JEM1400 electron microscope (JEOL, Akishima, Tokyo, Japan). The relative cytosolic areas in cross-sections of 10 cells were analyzed by Image-Pro Plus software. And fluorescence signals obtained using anti-FLAG (mouse) (A8592, Sigma), anti-FLAG (rabbit) (20543-1-AP, Proteintech), anti-HA (mouse) (H3663, Sigma), anti-HA (rabbit) (C29F4, Cellular signaling), anti-SC35 antibody (Ab11826, Abcam), and anti-SF2 antibody (sc-33652, Santa Cruz) were acquired and analyzed by Carl Zeiss LSM 880 microscope (Carl Zeiss, Germany). At least 10 cells from each group were analyzed.
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