Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Co-immunoprecipitation of endogenous SET8 and PARP1 in HCT116 cells. Rabbit IgG (left lane) was used as a negative control. (B) Colocalization between FLAG-PARP1 (red), GFP-SET8 (green) and endogenous PCNA (pink) in COS-7 cells. DAPI (blue) represents the nuclear DNA content. (C) Mapping of domain interactions using GST-pulldown assays between SET8 domains (top, left) and full-length recombinant PARP1 protein and GST-pulldown assays between PARP1 domains and full-length recombinant SET8 protein (top, right). The PARP1 or SET8 binding were detected by western blotting using PARP1 antibody (middle, left) or SET8 antibody (middle, right) respectively. Ponceau stain gels (bottom) represent the amount of GST beads constructs used for the GST-pulldown assays. (D) In vitro detection of full-length recombinant SET8 ADP-ribosylation by full-length recombinant PARP1 by western blotting using anti-ADP ribose antibody (top). Ponceau stain gels (bottom) represent the amount of PARP1 and SET8 recombinant enzyme used for ADP-ribosylation assay (bottom). (E) Detection of SET8 lysines ADP-ribosylation using mass spectrometry analysis of SET8 ADP-ribosylated peptides by full-length recombinant PARP1 protein in vitro .

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Immunoprecipitation, Negative Control, Recombinant, Binding Assay, Western Blot, Staining, Construct, In Vitro, Mass Spectrometry

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Detection of unbound 100 bp DNA ladder by TBE ethidium bromide-stained gel in supernatants (left lane) on GST-SET8 domains or mutant (M) using GST-pulldown assays (top, left side). Asterisks are representing shift of DNA on GST-SET8 157-352 amino acid protein or GST-SET8 full-length protein (FL) beads. Ponceau stain represents the amount of GST beads constructs used for the GST-pulldown (bottom, left side). (B) Different concentrations of recombinant full-length SET8 protein binding to DNA using EMSA to determine the equilibrium dissociation constant (Kd). (C) Detection of unbound mononucleosome by TBE ethidium bromide-stained gel in supernatants on GST beads versus GST-SET8 domains or mutant (M) beads using GST-pulldown assays. (D) Detection of unbound DNA (left side) or unbound mononucleosome (right side) by TBE ethidium bromide-stained gel in supernatants (top) on GST beads versus GST-SET8 FL beads using GST-pulldown assays. GST or GST-SET8 FL beads were poly ADP-ribosylated or not (with or without NAD) using full-length recombinant PARP1 as demonstrated by western blot using anti-ADP ribose antibody (middle). Ponceau stain gel (bottom) represents the amount of GST bead constructs used for the GST-pulldown and the ADP-ribosylation western blot analysis. (E) SET8 histone methyltransferase assay on full-length recombinant histone H4 using full-length recombinant SET8 in presence or absence of activated full-length recombinant PARP1.

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Staining, Mutagenesis, Construct, Recombinant, Protein Binding, Western Blot, HMT Assay

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells with or without FLAG-PARP1 overexpression in presence or not of the proteasome inhibitor MG132. Western blots detecting the amount of GFP-SET8 protein overexpressed in total extract (top, left) as well as the amount of FLAG-PARP1 protein overexpressed (top, right) using anti-GFP and anti-FLAG antibody respectively. Western blots detecting the amount of ubiquitin (Ub) (bottom, left) or ADP-ribosylation (bottom, right) of immunoprecipitated GFP-SET8 protein using anti-HA and anti-ADP ribose antibody respectively. Anti-actin was used as a loading control (middle). (B) GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells in presence or not (DMSO) of Cullin inhibitor (Cul4Ai) or PARG inhibitor (PARGi). Western blot detection of total GFP-SET8 protein levels overexpressed in COS-7 cells (top). Anti-actin antibody was used as control (middle). Western blot detecting the amount of poly-ADP ribosylation (bottom) of immunoprecipitated GFP-SET8 protein using an anti-ADP ribose antibody. (C) Western blots (left side) detecting the amount of PARP1 (top), SET8 protein (middle) as well as the amount of H4K20me1, H4K20me2 and H4K20me3 levels (bottom) in total protein extract of knockdown HeLa cells treated with esiRNA GFP (control), esiRNA PARP1 and esiRNA SET8, respectively. Respective densitometry analyses of protein abundance representative of at least 2 biological experiments are shown (right side). Ponceau stain was used as control (left side).

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Immunoprecipitation, Over Expression, Western Blot, Ubiquitin Proteomics, Control, Knockdown, esiRNA, Quantitative Proteomics, Staining

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Western blot indicating PARP1 (top, left), SET8 (middle, left) and H4K20me1 (middle, left) levels in total protein extracts from HeLa cells synchronized in G1, S and G2/M phases, respectively. Western blot of CDT1 protein levels is shown as a cell cycle synchronization control as well as Ponceau stain for loading control and densitometry analyses (bottom, left). Respective densitometry analyses of H4K20me1 (top, right) and SET8 (bottom, right) relative protein abundances are shown (right) and representative of at least 2 biological experiments. (B) SET8 immunoprecipitation from total protein extract in HeLa cells synchronized in G1, S and G2/M phases. Western blots detection of PARP1 (top, left) as well as SET8 immunoprecipitated protein levels (bottom, left) are revealed. Densitometry analyses of PARP1/SET8 ratio during G1, S and G2/M cell cycle phases are shown (right) and are representative of at least 2 biological experiments. (C) Pulsed chased cells with 5-ethynyl-2 -deoxyuridine (EdU) to label DNA (magenta) is transfected with GFP-SET8 (green). Endogenous ADP-ribose (red) is revealed by anti-ADP-ribose conjugated with Texas Red. Merged images demonstrates the co-localization of EDU, SET8 and ADP-ribose.

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Western Blot, Control, Staining, Immunoprecipitation, Transfection

Journal: NPJ Science of Food

Article Title: The Artemisia argyi oil reduces high-fat diet-induced obesity by enhancing thermogenesis in brown adipose tissue

doi: 10.1038/s41538-025-00633-2

Figure Lengend Snippet: A Western blot analysis showing the expression of UCP1, PGC1α, PPARα, PRDM16, and AGT in the BAT of mice from the NCD, HFD, LA, MA, and HA groups. B – F The mRNA levels of Ucp1 , Pgc1α , Pparα , Prdm16 , and Agt in the BAT of mice (n = 6). G Immunofluorescence (IF) analysis for the UCP1 expression in BAT of mice. Scale bar, 50 μm. H Protein expression levels of COXII and COXIV in BAT of mice. The mRNA levels of Cox8b ( I ) and Cox5b ( J ) in BAT were determined by qPCR analysis. * denote the level of statistical significance. ns no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test ( B – F , I – J ).

Article Snippet: Membranes were blocked with Fastest Blocking Reagent (HYCEZMBIO, Cat#HYC00811) and incubated with primary antibodies at the following dilutions: UCP1 (Diagbio, Cat#db9840) (dilution 1:1000), PRDM16 (Santa, Cat#sc-517625) (dilution 1:1000), PGC1α (SAB, Cat#37818) (dilution 1:1000), β-actin (Sino Biological, Cat#109444-T36) (dilution 1:20000), FASN (BOSTER, Cat#PB9865) (dilution 1:1000), PPARα (Biodragon, Cat#BD-PB4080) (dilution 1:1000), HSL (ZENBIO, Cat#344379) (dilution 1:1000), Flag (Sigma-Aldrich, Cat#F7425) (dilution 1:1000), HA (Bioss, Cat#BMS0966M) (dilution 1:1000), PPARγ (Bioworld, Cat#BS79617) (dilution 1:1000), ATGL (Abways, Cat#CY8408) (dilution 1:1000), ACC1 (HABIO, Cat#ET1609-77) (dilution 1:1000), IL10 (Solarbio, Cat#K009382P) (dilution 1:1000), TNFα (ELK Biotechnology, Cat#EA251) (dilution 1:1000), ZFP516 (GenScript) (dilution 1:1000), and LSD1 (PTM BIO, Cat#PTM-5960) (dilution 1:1000).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: NPJ Science of Food

Article Title: The Artemisia argyi oil reduces high-fat diet-induced obesity by enhancing thermogenesis in brown adipose tissue

doi: 10.1038/s41538-025-00633-2

Figure Lengend Snippet: A The diagram illustrates that the BAT SVFs were isolated and differentiated into mature brown adipocytes, followed by AAO treatment for western blot analysis, qPCR, and OCR analysis. B The TG content in the cells of each group. C The protein expression levels of UCP1, PRDM16, AGT, and PPARγ in the cells of each group. D – G The mRNA expression levels of Ucp1 , Prdm16 , Agt , and Pparγ . H – J BAT SVF cells were differentiated into brown adipocytes, followed by treatment with AAO. Oligomycin, FCCP, and Rotenone/Antimycin were added at the specified time points, indicated by the arrows, and the OCR data are shown in ( H ). The average basal and maximal respiration rates are presented in ( I ) and ( J ). * denote the level of statistical significance. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test ( B , D – G , I , J ).

Article Snippet: Membranes were blocked with Fastest Blocking Reagent (HYCEZMBIO, Cat#HYC00811) and incubated with primary antibodies at the following dilutions: UCP1 (Diagbio, Cat#db9840) (dilution 1:1000), PRDM16 (Santa, Cat#sc-517625) (dilution 1:1000), PGC1α (SAB, Cat#37818) (dilution 1:1000), β-actin (Sino Biological, Cat#109444-T36) (dilution 1:20000), FASN (BOSTER, Cat#PB9865) (dilution 1:1000), PPARα (Biodragon, Cat#BD-PB4080) (dilution 1:1000), HSL (ZENBIO, Cat#344379) (dilution 1:1000), Flag (Sigma-Aldrich, Cat#F7425) (dilution 1:1000), HA (Bioss, Cat#BMS0966M) (dilution 1:1000), PPARγ (Bioworld, Cat#BS79617) (dilution 1:1000), ATGL (Abways, Cat#CY8408) (dilution 1:1000), ACC1 (HABIO, Cat#ET1609-77) (dilution 1:1000), IL10 (Solarbio, Cat#K009382P) (dilution 1:1000), TNFα (ELK Biotechnology, Cat#EA251) (dilution 1:1000), ZFP516 (GenScript) (dilution 1:1000), and LSD1 (PTM BIO, Cat#PTM-5960) (dilution 1:1000).

Techniques: Isolation, Western Blot, Expressing

Journal: Journal of the Endocrine Society

Article Title: Validation of a new Custom Polyclonal Progesterone Receptor Antibody for Immunohistochemistry in the Female Mouse Brain

doi: 10.1210/jendso/bvad113

Figure Lengend Snippet: Immunoreactivity produced by commercial and custom PR antibodies in the arcuate nucleus. (A) Schematic diagram of the human PR-A and PR-B protein amino acid sequences, with the corresponding immunogen sequences of tested PR antibodies mapped. Host species of antibodies is indicated by r (rabbit) or m (mouse). AF, activation functions; DBD, DNA-binding domain; h, hinge region; LBD, ligand-binding domain; NTD, N-terminal domain. (B-H) Representative images of chromogenic immunohistochemical labeling in the arcuate nucleus of adult diestrous female mice produced by the (B) discontinued A0098 DAKO PR antibody, (C-G) the commercially available MA1-410 (1:5000), PA1413 (1:500), bs-0111R (1:1000), and MAB9785 (1:2000) PR antibodies, and (H) the newly generated custom RC269 PR antibody (1:2500) on paraformaldehyde-fixed brain sections. (F, G) Representative images of the high-signal (n = 3 mice) and low-signal (n = 3) MAB9785 PR antibody labeling observed across the 6 animals tested. Scale bar = 100 µm. 3 V, third ventricle; ARN, arcuate nucleus.

Article Snippet: PA1413 Boster Bio (Pleasanton, CA, USA) , Rabbit polyclonal , 536-553aa N-terminal domain (close to the DNA-binding domain) , AB_2923359 , Unclear , [ ] .

Techniques: Produced, Activation Assay, Binding Assay, Ligand Binding Assay, Immunohistochemical staining, Labeling, Generated, Antibody Labeling