Total RNA was isolated from different cells as described. The m 6 A dot blot assay was performed following a published protocol [12] . Briefly, the RNA samples in certain volume were loaded to the PVDF membrane (IPVH0010, Milipore) and Ultraviolet crosslinker was used to crosslink RNA and membrane. Then the membrane was blocked and incubated with anti-m 6 A antibody (1:1000 dilution, ab208577, Abcam) overnight at 4 °C. Then the membrane was incubated with secondary antibody at room temperature, and the membrane was detected with ECL Detection Reagent (PK10002, Proteintech). The signal of each dot was tested by Minichem TM Chemiluminescence Imaging System (Sagecreation, Beijing, China) in all experiments. The m 6 A RNA methylation quantification kit (p-9005, EpigenTek) was used to detect the m 6 A content. Two hundred nanogram RNAs were added to the wells. We conducted RNA m 6 A quantification assay following the manufacturer's protocol. The m 6 A content was quantified by measuring the absorbance at 450 nm.
Ecl detection reagent
Manufactured by Proteintech
Sourced in China
The ECL Detection Reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It generates a luminescent signal when exposed to horseradish peroxidase (HRP)-conjugated detection antibodies, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ab208577, by Abcam (1 mentions)
M6a rna methylation quantification kit, by Epigentek (1 mentions)
Ipvh0010, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti ulk1, by Cell Signaling Technology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti caspase 1, by Proteintech (1 mentions)
Anti histone h3, by Immunoway (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Anti p50, by Cell Signaling Technology (1 mentions)
4 protocols using ecl detection reagent
1
m6A RNA Methylation Detection Protocol
2
Comprehensive Protein Analysis Protocol
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In our study, total protein was extracted with lysis buffer (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, 0.5% sodium deoxycholate, and 1% NP-40), and the supernatant was then collected by centrifugation. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk in TBS containing 0.05% Tween-20 (TBST), incubated with primary antibodies overnight at 4°C, washed with TBST, and incubated with the secondary antibody (DyLight 488-conjugated mouse anti-rabbit IgG; 1:5000). Protein expression was then detected using an ECL detection reagent (Proteintech, Hubei, China). In this analysis, the following primary antibodies were used: anti-LACTB, anti-E-cadherin, anti-vimentin, anti-N-cadherin, anti-Twist1, anti-PI3K, anti-PIK3R3, anti-AKT, anti-p-AKT, anti-mTOR, anti-4E-BP1, anti-LC3, anti-P62, anti-C-Myc, anti-cyclinD1, and anti-ULK1 (Cell Signaling Technology, USA). An anti-β-actin antibody (Santa Cruz Biotechnology, USA) was used as a loading control.
3
Protein Expression Analysis by Western Blot
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Equal amounts of proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Darmstadt, Germany). The membranes were incubated with 5% buttermilk for 1 h at 25°C and then incubated with primary antibodies at 4°C overnight with gentle shaking. The primary antibodies included anti-N-cadherin (ab76011), anti-Vimentin (ab92547), anti-cit-H3 (ab5103), (1:1000, Abcam, UK), anti-NLRP3 (19771-1-AP), anti-E-cadherin (20874-1-AP), anti-Caspase1 (22915-1-AP), anti-GAPDH (60004-1-Ig) (1:1000, 1:5000, 1:1000, and 1:20000 Proteintech, China), anti-p50 (1:1000, Cell Signaling Technologies, 13586, USA), anti-p-p50 (sc-271908), anti-p65 (sc-8008), anti-p-p65 (sc-136548) (all at 1:500, Santa Cruz Biotechnology, USA), and anti-Histone H3 (1:1000, ImmunoWay Biotechnology, YT2163, USA). The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies specific for rabbit or mouse primary antibodies (Proteintech, China) at a dilution of 1:2000 at 25°C for 1 h. The protein bands were detected utilizing ECL Detection Reagents (Proteintech, China).
4
Western Blot Analysis of Protein Expression
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Western blotting analysis was performed as described previously (18 (link)). Total proteins were isolated using the RIPA buffer [50 mM Tris pH 8.0, 1% NP-40, 150 mM NaCl, 0.1% sodium dodecylsulfate (SDS) and 1 mM ethylenediaminetetraacetic acid (EDTA)] containing 0.5 mM phenylmethanesulfonylfluoride (Beyotime, China). The protein concentration was determined using the BCA protein assay kit (Beyotime, China) following the manufacturer’s instructions. Equal amounts of proteins were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and then electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Subsequently, the membranes were blocked with 5% nonfat dry milk (Bio-rad, USA) in Tris-buffered saline (TBS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) containing 0.05% Tween-20 for 1 h, followed by incubation with primary antibodies against WTAP (1:1,000), CDK1 (1:4,000), BAX (1:2,000), Mcl-1 (1:1,000), Wee1 (1:1,000), GSK-3β (1:1,000), GSK-3β pSer9 (1:1,000), H2A (1:1,000), GAPDH (1:1,000), or β-actin (1:1,000). Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000) at room temperature for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) detection reagents (ProteinTech, USA).
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