Frozen liver tissues were dounce homogenized in RIPA buffer. After incubating on ice for 10 min, homogenates were centrifuged at full speed for 15 min at 4°; supernatant was then collected and stored at −80°. Protein was quantified with a BCA assay (Thermo Scientific) and 2 μg/μL lysates were boiled for 5 min in 5x loading buffer. Denatured protein lysates were loaded in precast polyacrylamide gels (BioRad) and transferred to PVDF membranes (BioRad). Membranes were blocked with 5% milk in PBST and probed with primary antibodies for BCL6 (Santa Cruz, D-8) at 1:200, PPARα (Santa Cruz, H-98) 1:500, pAKT (Cell Signaling, 4060) 1:1000, panAKT (Cell Signaling, 4691) 1:1000 or β-actin (Sigma, A1978) 1:1000 overnight at 4°. Secondary antibodies were added for 1 hr at room temperature (Jackson ImmunoResearch). Protein was then visualized using ECL (ThermoScientific). MemCode Reversible Stain was used to visualize total protein (Thermo Fisher Scientific). Protein densitometry was quantified using ImageJ 1.51 s (Schneider et al., 2012 (link)).
Memcode reversible stain
Manufactured by Thermo Fisher Scientific
The MemCode Reversible Stain is a laboratory reagent used for the detection and visualization of proteins in polyacrylamide gels. It is a reversible staining technique that allows for the subsequent analysis and identification of specific proteins of interest.
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Lab products found in correlation
S adenosyl l methyl 3h methionine, by PerkinElmer (4 mentions)
Ready gel, by Bio-Rad (2 mentions)
En3hance spray surface autoradiography enhancer, by PerkinElmer (2 mentions)
Precast polyacrylamide gel, by Bio-Rad (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Pparα, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Tris tricine precast gel, by Bio-Rad (1 mentions)
Anti γh2ax antibody, by Merck Group (1 mentions)
Cellytic m cell lysis reagent, by Merck Group (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
11 protocols using memcode reversible stain
1
Western Blot Analysis of Liver Proteins
2
Methylation Assay for PTEN by SMYD2
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In vitro methyltransferase assays were described previously [17,19–22] . Briefly, recombinant PTEN protein was incubated with recombinant SMYD2 and 2 μCi S-adenosyl-l -[methyl-3H]-methionine (PerkinElmer, Waltham, MA) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT, and 10 mM MgCl2) for 1 hour at 30°C. After denaturing, samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), blotted to polyvinylidene difluoride membrane, and visualized by MemCode Reversible Stain (Thermo Fisher Scientific, Waltham, MA) and fluorography.
3
Doxorubicin-Induced Phosphorylation of H2AX
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HeLa cells were lysed with CelLytic M cell lysis reagent (Sigma-Aldrich) containing protease and phosphatase inhibitors 2 h after treatment with 1 μM of doxorubicin and used as enzyme source. Biotin-tagged unmethylated H2AX peptide (Biotin-SATVGPKAPSGGKKATQASQEY) or biotin-tagged methylated H2AX peptide (Biotin-SATVGPKAPSGGK[me2K]ATQASQEY) is mixed with different amount of doxorubicin-induced HeLa cell lysates in the kinase buffer (50 mM HEPES, pH 7.5, 50 mM potassium chloride, 5 mM magnesium chloride, 10% glycerol, 1 mM ATP and 1 mM dithiothreitol) for 90 min at 30 °C. Peptides were precipitated with Dynabeads M-280 streptavidin (Life Technologies) and the samples were separated on SDS–PAGE using the Tris-Tricine Precast Gel (456-3063, Bio-Rad). Subsequently, WB was performed using an anti-γ-H2AX antibody (Millipore). The nitrocellulose membrane for the WB was stained with MemCode Reversible Stain (Thermo Scientific) to quantify the amount of peptides.
4
In Vitro Methyltransferase Assay of EGFR
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For the in vitro methyltransferase assay, recombinant EGFR (0.3 μg/μL, 3.6 μM, Life Technologies, 90.5 kDa, #PV3872) was incubated with recombinant WHSC1L1 enzyme (BPS Bioscience, 0.62 μg/μL, 10.2 μM, 65 kDa) using 1 mCi S-adenosyl-L-[methyl-3H]-methionine (SAM; PerkinElmer) as the methyl donor in a mixture of 30 μL of methylase activity buffer (50 mM Tris-HCl at pH8.8, 10 mM dithiothreitol and 10 mM MgCl2) overnight at 30 °C. Proteins were separated on a 5–20% SDS-PAGE gel (Ready Gel; Bio-Rad), then transferred on a PVDF membrane and visualized by MemCode Reversible Stain (Thermo Scientific) and fluorography.
5
Subcellular Fractionation and Western Blot Analysis
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Subcellular fractionations were carried out as previously described with modifications.40 The purity of each fraction was further validated by using anti‐rabbit TLR‐4 (Santa Cruz, sc‐10741; membrane marker), anti‐rabbit Caspase‐3 (Cell Signaling, 9662S; cytosolic marker), and anti‐rabbit Histone H3 (Cell Signaling, 4499s; nuclear marker).41 Western blot was performed on flash snap‐frozen human myocardium tissues as we previously published.40 , 41 The below primary antibodies were used: anti‐rabbit TFR‐1 (Cell Signaling, 13208s); anti‐rabbit FPN (Novus, NBP1‐21502); anti‐rabbit FTN (Abcam, ab75973); anti‐mouse DMT‐1 (Abcam, ab55735), followed by incubation with HRP‐conjugated secondary antibodies at 1/5000 dilution (Cell Signaling). The total protein loadings were visualized by MemCode reversible stain (24585, Thermo Scientific) as a loading control. Fiji ImageJ software (NIH, Bethesda, MD) was used for band intensity quantitation.
6
In vitro PRMT6 Methyltransferase Assay
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In vitro methyltransferase assays were described previously [29 , 30 ]. Briefly, recombinant p21 protein was incubated with recombinant PRMT6 protein and 2 μCi S-adenosyl-L-[methyl-3H]-methionine (Perkin Elmer) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT and 10 mM MgCl2) for 1 h at 30°C. After denaturing, samples were separated by SDS-PAGE, blotted to PVDF membrane and visualized by MemCode Reversible Stain (Thermo Fisher Scientific) and fluorography.
7
In vitro Methyltransferase Assay for β-catenin
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In vitro methyltransferase assays were performed as described previously [19 (link), 20 (link)]. Briefly, recombinant GST-WT-β-catenin protein (#12-537; Millipore) was incubated with recombinant His-SMYD2 protein (in-house) and 2 μCi S-adenosyl-L- [methyl-3H]-methionine (Perkin Elmer, Waltham, MA) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT and 10 mM MgCl2) for 2 h at 30°C. After denaturing, samples were separated by SDS-PAGE, blotted to PVDF membrane and visualized by MemCode Reversible Stain (Thermo Fisher Scientific, Waltham, MA) and fluorography.
8
In Vitro Histone H2AX Methylation Assay
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For the in vitro methyltransferase assay, recombinant Histone H2AX (#14-576, Millipore) was incubated with recombinant SUV39H2 enzyme using 1 μCi S-adenosyl-L -[methyl-3H]-methionine (SAM; PerkinElmer) as the methyl donor in a mixture of 10 μl of methylase activity buffer (50 mM Tris-HCl at pH8.8, 10 mM dithiothreitol and 10 mM MgCl2), for 2 h at 30 °C48 (link)49 (link)50 (link)51 . Proteins were resolved on a 5–20 % SDS–PAGE gel (Ready Gel; Bio-Rad) and visualized by MemCode Reversible Stain (Thermo Scientific) and fluorography.
9
Methyltransferase Assay for SUV420H1 and ERK
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In vitro methyltransferase assays were described previously [45 (link)–49 (link)]. Briefly, recombinant SUV420H1 protein was incubated with recombinant ERK1 and 2 μCi S-adenosyl-L-[methyl-3H]-methionine (PerkinElmer) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT, and 10 mM MgCl2) for 1 hour at 30°C. After denaturing, samples were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), blotted to polyviny- lidene difluoride membrane, and visualized by MemCode Reversible Stain (Thermo Fisher Scientific) and fluorography.
10
In vitro Methyltransferase Assay for AKT1
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For the in vitro methyltransferase assay, recombinant AKT1 (P2999, Thermo Fisher Scientific) was incubated with recombinant SMYD3 enzyme using 2 μCi S-adenosyl-L-[methyl-3H]-methionine (SAM; PerkinElmer) as the methyl donor in a mixture of 10 μl of methylase activity buffer (50 mM Tris-HCl at pH8.8, 10 mM dithiothreitol (DTT) and 10 mM MgCl2), for 2 hours at 30°C [37 (link)–46 (link)]. Proteins were resolved on a Mini-PROTEAN® TGX™ Precast gel (Any kD; Bio-Rad) and visualized by fluorography using EN3HANCE™ Spray Surface Autoradiography Enhancer (PerkinElmer). Loading proteins were visualized by MemCode™ Reversible Stain (Thermo Fisher Scientific).
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