primary antibodies against t- bet, gata3, dnmt1, βactin, foxo3, p- p65, p65 and laminb  (Proteintech)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Homocysteine accelerates hepatocyte autophagy by upregulating TFEB via DNMT3b-mediated DNA hypomethylation

doi: 10.3724/abbs.2023060

Figure Lengend Snippet: The effect of DNMT3b on TFEB DNA methylation and autophagy (A) The difference in TFEB mRNA and protein levels in hepatocytes exposed to Hcy and DC-05, TF-3, and nanomycin A (NA), the corresponding inhibitors of DNMT1, DNMT3a and DNMT3b, respectively. (B) The binding level between DNMT and the TFEB promoter in hepatocytes was examined by ChIP assay. (C) DNMT3b mRNA and protein levels were measured by qRT-PCR and western blot analysis, respectively in hepatocytes with DNMT3b adenovirus infection. (D) DNMT3b levels were measured by qRT‒PCR and western blot analysis in hepatocytes infected with sh-DNMT3b adenovirus (sh-DNMT3b-1, sh-DNMT3b-2, and sh-DNMT3b-3). (E) Quantitative analysis of DNA methylation of the TFEB promoter in hepatocytes after DNMT3b overexpression or inhibition. (F) The effect of DNMT3b overexpression on TFEB level in hepatocytes. (G) The binding level of DNMT3b to the TFEB promoter was measured by ChIP assay in hepatocytes exposed to Ad-DNMT3b. (H) Detection of LC3BII/I and p62 protein levels after overexpression of DNMT3b. (I) Confocal images of mRFP-GFP-LC3 fluorescent puncta in hepatocytes after exposure to Ad-DNMT3b (scale bar: 5 μm). Data are presented as the mean±SD from three independent experiments, * P<0.05, ** P<0.01.

Article Snippet: Membranes were blocked with 5% defatted milk and then incubated with primary antibodies against DNMT1 (PA5-30581; Thermo Fisher Scientific), TFEB (ab267351; Abcam, Cambridge, USA), DNMT3a (ab2850; Abcam), DNMT3b (ab2851; Abcam), p62 (ab109012; Abcam), microtubule-associated protein 1 light chain 3 (LC3; ab192890; Abcam), and β-actin (ab8226; Abcam) respectively, followed by incubation with horseradish peroxidase (HRP)-labelled secondary antibody.

Techniques: DNA Methylation Assay, Binding Assay, Quantitative RT-PCR, Western Blot, Infection, Over Expression, Inhibition

Journal: Virology journal

Article Title: Vpr driving DNA methylation variation of CD4 + T cells in HIV-1 infection.

doi: 10.1186/s12985-024-02363-5

Figure Lengend Snippet: Fig. 5 The impact of Vpr on DNA methylation of host cell is verified by western blot. (A, B) The effect of Vpr on differentially methylated cytosines (DMCs) in the promoter (A) or gene-body (B) region of DNMTs. (C) The effect of Vpr on protein levels of DNMT1 and DNMT3A. (D, E) The effect of Vpr on DMCs in the promoter (A) or gene-body (B) region of CTCF and PAX5. (F) The effect of Vpr on protein levels of CTCF and PAX5.

Article Snippet: Primary antibodies against DNMT1 (24206-1-AP) and DNMT3A (20954-1-AP), were purchased from proteintech Group (Wuhan, China).

Techniques: DNA Methylation Assay, Western Blot, Methylation

Journal: FEBS Open Bio

Article Title: Epigenome‐wide DNA methylation analysis of myasthenia gravis

doi: 10.1002/2211-5463.13656

Figure Lengend Snippet: (A) Protein level of DNMT1 expression in HT22 cell line. (B) Analyses of the protein level of DNMT1 expression in HT22 cell line normalized to those of β‐actin for each sample. **** P < 0.0001 versus the sham group. n = 4. The data are presented as mean ± standard deviation (SD). A one‐way ANOVA followed by Fisher's least significant difference was used for statistics.

Article Snippet: Primary antibodies against DNMT1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Standard Deviation

Journal: Leukemia

Article Title: Inhibition of SUMOylation enhances DNA hypomethylating drug efficacy to reduce outgrowth of hematopoietic malignancies

doi: 10.1038/s41375-023-01838-8

Figure Lengend Snippet: A Mechanistic model for combining the drugs 5-Aza-2’ and TAK981. 5-Aza-2’ incorporates into the DNA at the site of cytidine. DNMT1 binding to 5-Aza-2’ gets trapped and is subsequently massively SUMOylated, ubiquitinated and degraded by the proteasome. Upon 5-Aza-2’and TAK981 treatment, DNMT1 remains entrapped at the DNA. B Namalwa cells were cultured in suspension and treated for 4 or 20 h with 1 µM 5-Aza-2’ and/or 1 µM TAK981 or DMSO 0.1% as control with or without MG132 10 µM for 4 h and 2.5 µM for 20 h. Total lysates were analyzed by immunoblotting using antibodies directed against DNMT1 and SUMO2/3. PonceauS staining was used as control. C DNMT1 foci were visualized by confocal microscopy. Namalwa cells were treated for 4 or 20 h with 1 µM 5-Aza-2’ and/or 1 µM TAK981 or DMSO 0.1% as control and cells were spun onto glass coverslips and stained. Representative images are depicted. Scale bars represent 10 µm. D Quantification of images from C . The graph depicts DNMT1 foci. Dots represent the numbers of DNMT1 foci/cell. 100 cells per replicate were analyzed ( n = 3). P-value ** ≤ 0.01, *** ≤ 0.005. One-way ANOVA was performed with Graphpad Version 9.3.1. E , F Respectively show Ni-NTA pulldown of His10-SUMO2- and His10-ubiquitin. Namalwa cells were cultured in suspension and treated for 4 or 20 h with 1 µM 5-Aza-2’ and/or 1 µM TAK981 or DMSO 0.1% as control with or without MG132 10 µM for 4 h and 2.5 µM for 20 h. Total lysates (TL) and elutions from His10 pulldowns (PD) were analyzed by immunoblotting with antibodies directed against DNMT1, SUMO2/3 or ubiquitin. Equal loading was verified with Ponceau S staining.

Article Snippet: Primary antibodies against DNMT1 (rabbit, 1:1000, 5032 S, Cell Signaling Technology, Leiden, NL), SUMO2/3 (1:500, mouse monoclonal 8A2, University of Iowa), ubiquitin (1:5000, sc8017, Santa Cruz, Dallas, TX, USA) were incubated with membrane in 5% milk powder in PBS − 0.05% Tween20.

Techniques: Binding Assay, Cell Culture, Suspension, Control, Western Blot, Staining, Confocal Microscopy, Ubiquitin Proteomics

Journal: Leukemia

Article Title: Inhibition of SUMOylation enhances DNA hypomethylating drug efficacy to reduce outgrowth of hematopoietic malignancies

doi: 10.1038/s41375-023-01838-8

Figure Lengend Snippet: A γH2Ax and DNMT1 foci visualized with confocal microscopy. U2OS cells cultured on glass cover slips were treated with 1 µM 5-Aza-2’ and/or 1 µM TAK981 or DMSO 0.1% as control for 4 or 20 h. Slides were stained with DNMT1 and γH2Ax antibodies. The panel shows representative images of each condition, single DNMT1 stain, single γH2Ax stain and a merged image including Hoechst staining. Scale bar represents 25 µm B γH2Ax foci quantification of images from A . Dots represent numbers of γH2Ax foci/cell using ~100 cells per replicate ( n = 3). P-value * ≤ 0.05. One-way ANOVA was used, followed by Tukey’s multiple comparisons with Graphpad prism Version 9.3.1. C BRCA1 knock out in RPE-1 p53 -/- cells was validated via immunoblotting. PonceauS staining was used as loading control. D Colony formation analysis of homologous recombination (HR) deficient cell line RPE-1 p53 -/- BRCA1 -/- vs RPE-1 p53 -/- 2,500 cells were seeded per well in 6-well plates, treated with 5-Aza-2’ and/or TAK981 at the indicated doses and grown for 14 days. Subsequently, cells were fixed, stained with crystal violet and quantified. P-value * ≤ 0.05, ** ≤ 0.01. Two-sided t -test RPE-1 p53 -/- BRCA1 -/- vs RPE-1 p53 -/- per treatment condition with Graphpad Version 9.3.1. E Ni-NTA pulldown of His10-SUMO2- for validation of targets from mass spectrometry analysis (Fig. ). U2OS cells were cultured in suspension and treated for 4 or 20 h with 1 µM 5-Aza-2’ or DMSO 0.1% control with or without MG132 10 µM for 4 h and 2.5 µM for 20 h. Total lysate input and His10-SUMO2- pulldown elutions were analyzed by immunoblotting using SMC6 antibody. Equal loading was verified with Ponceau S staining. F SMC6 foci were identified via confocal microscopy upon treatment with 1 µM 5-Aza-2’ and/or 1 µM TAK981 for 4 or 20 h and quantified. Dots represent numbers of SMC6 foci/cell using ~50–100 cells per replicate ( n = 3). P-value * ≤ 0.05, ** ≤ 0.01. One-way ANOVA, Tukey’s multiple comparisons with Graphpad prism Version 9.3.1.

Article Snippet: Primary antibodies against DNMT1 (rabbit, 1:1000, 5032 S, Cell Signaling Technology, Leiden, NL), SUMO2/3 (1:500, mouse monoclonal 8A2, University of Iowa), ubiquitin (1:5000, sc8017, Santa Cruz, Dallas, TX, USA) were incubated with membrane in 5% milk powder in PBS − 0.05% Tween20.

Techniques: Confocal Microscopy, Cell Culture, Control, Staining, Knock-Out, Western Blot, Homologous Recombination, Biomarker Discovery, Mass Spectrometry, Suspension

Journal: Leukemia

Article Title: Inhibition of SUMOylation enhances DNA hypomethylating drug efficacy to reduce outgrowth of hematopoietic malignancies

doi: 10.1038/s41375-023-01838-8

Figure Lengend Snippet: A A panel of ten lymphoma cell lines was treated with TAK981 at a dose range of 0.01–1 µM and cell viability was measured after 4 days. Cells were divided over two graphs respectively: Burkitt Lymphoma cell lines and DLBCL cell lines. The graphs show cell viability in ratio to control per cell line. B The panel of ten lymphoma cell lines was treated with 5-Aza-2’ at a dose range of 0.01–10 µM and cell viability was measured after 4 days. Cells were divided over two graphs respectively, representing Burkitt Lymphoma cell lines or DLBCL cell lines. The graphs show cell viability in ratio to control per cell line. C IC50 values of the cell line panel for TAK981 0.00001–1 µM dose response and 5-Aza-2’ dose response 0.01–10 µM. IC50 values were calculated in Graphad Version 9.3.1. and MYC status for all cell lines ‘+’ indicates translocation of MYC gene (Table ), ‘-‘ represents no change in MYC gene (Table ). D The panel of ten lymphoma cell lines was treated with 5-Aza-2’ at the indicated dose range with or without 25 nM TAK981. Viability dose response curves were plotted individually per cell line. E Excess overbliss (%) of plots in D was calculated as detailed in methods section and visualized in a heat map (Higher % excess overbliss represents more synergy) for each dose of 5-Aza-2’ versus the same dose with 25 nM TAK981. F Total lysates of the panel of ten lymphoma cell lines were analyzed for protein expression levels of DNMT1, UBA2, MYC, UBC9, SUMO2/3 and SUMO1 by immunoblotting. γTubulin staining was used as a control. Immunoblotting of representative image of a total of n = 3 is shown. G Correlation of IC50 value and average excess overbliss percentage per cell line vs protein expression of DNMT1, UBC9 and MYC. Correlation was calculated in Graphpad Prism 9.3.1. Pearson r and P-value . Correlation data of the remainder of the proteins is depicted in Supplementary Fig. .

Article Snippet: Primary antibodies against DNMT1 (rabbit, 1:1000, 5032 S, Cell Signaling Technology, Leiden, NL), SUMO2/3 (1:500, mouse monoclonal 8A2, University of Iowa), ubiquitin (1:5000, sc8017, Santa Cruz, Dallas, TX, USA) were incubated with membrane in 5% milk powder in PBS − 0.05% Tween20.

Techniques: Control, Translocation Assay, Expressing, Western Blot, Staining