Journal: International Journal of Biological Sciences

Article Title: Identification of Histone Modifications Reveals a Role of H2b Monoubiquitination in Transcriptional Regulation of dmrt1 in Monopterus albus

doi: 10.7150/ijbs.59347

Figure Lengend Snippet: Gonadal histology and identification of histone modifications by LC-MS/MS. (A) Histological analysis of ovary, ovotestis, and testis by H&E. staining. O, ovary; Odg, degrading follicles; T, testis. Scale bar, 50 µm. Histones were extracted using acid extraction, resolved by 12 % Tricine-SDS-PAGE, and stained with Coomassie blue. The bands containing histones were cut into pieces based on size and digested by trypsin. The peptide segments were recovered and enriched by C18 column, and the peptide modifications were analyzed by LC-MS/MS. (B) Left panel indicates total number of H2a, H2b, H3, and H4 modification sites, including acetylation, methylation, and ubiquitination, among ovary, ovotestis, and testis. Right panel shows relative levels of modified sites normalized by total peptides with the sites, including acetylation, methylation, and ubiquitination, among ovary, ovotestis, and testis. (C) Mass spectrometry of H3K18 acetylation or dimethylation in the peptide covering residues 18-26 of histone H3 (KQLATKAAR) isolated from gonads. Top panel, annotated MS/MS spectrum of acetylation at H3K18 and K23 in ovary. Middle, annotated MS/MS spectrum of dimethylation at H3K18 in ovotestis. Bottom, annotated MS/MS spectrum of acetylation at H3K18 and K23 in testis. b: N-terminal fragment ion series; y: C-terminal fragment ion series.

Article Snippet: The primary antibodies were as follows: Rabbit antibody to Ubiquityl-Histone H2b (Lys 120) (Cat# 5546s) was purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, SDS Page, Modification, Methylation, Mass Spectrometry, Isolation, Tandem Mass Spectroscopy

Journal: International Journal of Biological Sciences

Article Title: Identification of Histone Modifications Reveals a Role of H2b Monoubiquitination in Transcriptional Regulation of dmrt1 in Monopterus albus

doi: 10.7150/ijbs.59347

Figure Lengend Snippet: Identification of post-translational modification sites on histones among ovary, ovotestis, and testis. (A) Distribution of histone acetylation sites on histones H2a, H2b, H3, and H4 in gonads. (B) Methylation sites identified on H2a, H2b, H3, and H4 in gonads. (C) Ubiquitination sites on H2a, H2b, H3, and H4 in gonads. Symbol key: the number represents the amino acid site of the peptide; the K is for lysine while the R for arginine; the blue line represents “testis”; the green line indicates “ovotestis”; the red line denotes “ovary”; rhombus is for acetylation; squares with 1, 2 and 3 are for mono-, di- and tri-methylation, respectively; circle is for ubiquitination; (D) Upper panel indicates the number of acetylation, methylation and, ubiquitination modification sites on H2a, H2b, H3, and H4 among ovary, ovotestis, and testis, respectively. Lower panel shows relative levels of modified sites normalized by total peptides with the sites, including acetylation, methylation, and ubiquitination, among ovary, ovotestis, and testis, respectively.

Article Snippet: The primary antibodies were as follows: Rabbit antibody to Ubiquityl-Histone H2b (Lys 120) (Cat# 5546s) was purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Modification, Methylation

Journal: International Journal of Biological Sciences

Article Title: Identification of Histone Modifications Reveals a Role of H2b Monoubiquitination in Transcriptional Regulation of dmrt1 in Monopterus albus

doi: 10.7150/ijbs.59347

Figure Lengend Snippet: Various modification sites at histone tails and in histone-fold domains among gonadal tissues. (A) Distribution of modifications at the N-ter histone tails, extended from the globular core of histones H2a, H2b, H3, and H4. DNA is wrapped around the nucleosome octamer made up of two H2a-H2b dimers and a H3-H4 tetramer. (B) Distribution of modification sites in histone-fold domains of H2a, H2b, H3, and H4. Post-translational modifications: acetylation (rhombus), methylation (squares with 1, 2, and 3 represent mono-, di- and trimethylation, respectively) and ubiquitination (circle). The numbers represent the amino acid sites in the peptide; the blue line, testis; the green line, ovotestis; the red line ovary; Blue R, arginine residues; Red K, lysine residues.

Article Snippet: The primary antibodies were as follows: Rabbit antibody to Ubiquityl-Histone H2b (Lys 120) (Cat# 5546s) was purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Modification, Methylation

Journal: International Journal of Biological Sciences

Article Title: Identification of Histone Modifications Reveals a Role of H2b Monoubiquitination in Transcriptional Regulation of dmrt1 in Monopterus albus

doi: 10.7150/ijbs.59347

Figure Lengend Snippet: Ubiquitylated H2b at K120 associated with spermatogenesis. (A) Mass spectrum of H2b peptide showing H2bK120 ubiquitylation. m/z, mass/charge ratio. (B) Ubiquitylation levels at H2b (K120) in ovary, ovotestis, and testis. The ratio of modified peptides to unmodified peptides are indicated. (C) Western blot analysis of the histone ubiquitylation level using the ubiquitylation-histone H2b (K120) antibody. Total protein samples were isolated from ovary, ovotestis, and testis. H2b or H3 were used as an internal control, respectively. (D) Immunofluorescent analysis of H2b in ovary, ovotestis and testis using anti-H2b antibody. (E) Immunofluorescent analysis of ubiquitylated histone H2b at K120 in ovary, ovotestis and testis using anti-ubiquitylated histone H2b at K120 antibody. The nuclei were stained by Hoechst (blue). The enlarged image originated from the region with white square. Sn, Sertoli cells; Sg, spermatogonia; Sc, spermatocytes; Tc, theca cells; Gc, granulosa cells; St, spermatids. Odg, degrading follicles; T, testis. Scale bar, 5 µm.

Article Snippet: The primary antibodies were as follows: Rabbit antibody to Ubiquityl-Histone H2b (Lys 120) (Cat# 5546s) was purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Modification, Western Blot, Isolation, Staining

Journal: Molecular Cancer

Article Title: Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

doi: 10.1186/1476-4598-13-116

Figure Lengend Snippet: Validation of nutlin-induced modulation of acetylated proteins in the AML cell line MOLM-13. (A) Western blots of total lysates from SILAC (stable labeling with amino acids in cell culture) labeled MOLM-13 cells treated with DMSO or 6 μM nutlin-3 for 6 hours using antibodies against p53, MDM2, Histone H2B, acetylated Histone H2B (Lys120), Hsp27, Hsp90, acetylated Hsp90 (Lys294) and actin. Bands were quantified using region of interest imaging analysis, and values are given as fold induction of control relative to actin. (B) MOLM-13 cells were treated nutlin-3 as described above, and immunoprecipitations using anti-acetyl lysine antibody or an unspecific antibody (rabbit IgG) were performed. Western blots of Hsp27 of the immunoprecipitated proteins and of the total lysates used in immunoprecipitations are shown. (C) Total levels of Hsp90 and Hsp27 in MOLM-13 cells treated with nutlin-3 as described above analyzed by flow cytometry. Results are given as representative flow diagrams and median fluorescence intensity (MFI) relative to control.

Article Snippet: The following antibodies were used; p53 (Bp53-12), Mdm2 (SMP-14) (Santa Cruz Biotechnology, CA, USA), Mdm2 (2A10), Mdm2 (IF2), anti-Hsp27 (G3.1) (Calbiochem, San Diego, CA, USA), p21 (SX118) (BD Biosciences, San Jose, CA, USA), phospho-p53 (Ser15), phospho-p53 (Ser20), ac-p53 (Lys382) (Cell Signaling Technologies, Beverly, MA, USA), anti-Histone H2B, anti-Hsp90 (Millipore, Temecula, CA, USA), anti-acetyl-Histone H2B (Lys120) (Upstate cell signaling solutions, Lake Placid, NY, USA), anti-acetyl-Hsp90 (Lys294) (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA), secondary horse radish peroxidase conjugated mouse and rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA), actin (AC-15) (Abcam plc, Cambridge, UK).

Techniques: Western Blot, Labeling, Cell Culture, Imaging, Immunoprecipitation, Flow Cytometry, Fluorescence

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: Antibody dilutions used.

Article Snippet: Overall, these results demonstrate that the anti-histone H2BK120 ubiquityl antibody (clone D11, Cell Signaling Technology) specifically recognizes and can be used to quantify histone H2BK119/K123 ubiquitination in budding as well as fission yeast cells.

Techniques:

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: ( a ) Sections of membrane probed with the indicated antibodies to quantify histones H3 or H2B and H2BK123ub1 levels in wild-type (WT) and indicated deubiquitinase mutant S. cerevisiae strains. The antibody dilutions used are listed in . ( b ) Quantitation of histones H3 (i) and H2BK123ub1 (ii) levels normalized to Ponceau S stained proteins from two or three independent experiments, respectively. ( c ) Sections of membrane probed with anti-H2B antibody (1:1000 dilution) (upper), anti-ubiquityl H2BK120 antibody (1:1000) (middle), and stained with Ponceau S (lower) using extracts prepared from S. cerevisiae strains expressing untagged or C-terminal V5 epitope-tagged histone H2B.

Article Snippet: Overall, these results demonstrate that the anti-histone H2BK120 ubiquityl antibody (clone D11, Cell Signaling Technology) specifically recognizes and can be used to quantify histone H2BK119/K123 ubiquitination in budding as well as fission yeast cells.

Techniques: Mutagenesis, Quantitation Assay, Staining, Expressing

Journal: Methods and Protocols

Article Title: Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting

doi: 10.3390/mps5050074

Figure Lengend Snippet: ( a ) Samples from extracts prepared from S. pombe strains expressing untagged H2B or Flag-tagged H2B were spearated and transferred onto a PVDF membrane. Shown are whole or sections of membrane probed with anti-histone H2B antibody from Active Motif and from Gene Tex and with anti-H2BK120 ubiquityl antibody. Ponceau S dye-stained sections used for normalization are also shown. Asterisk indicates cross-reacting proteins. ( b ) Samples from extracts prepared from wild-type S. pombe or strains lacking Ubp8 or Brl1 were separated and transferred to PVDF membrane. Shown are sections of membrane probed with anti-H2B (Gene Tex) or anti-H2BK120 ubiquityl antibody are shown. A section of the membrane stained with Ponceau S used for normalization is also shown. ( c ) Quantitation of H2BK119ub1 levels in the S. pombe ubp8-null mutant relative to wild-type (WT) strain. Error bars denote standard deviation of the mean from three independent experiments.

Article Snippet: Overall, these results demonstrate that the anti-histone H2BK120 ubiquityl antibody (clone D11, Cell Signaling Technology) specifically recognizes and can be used to quantify histone H2BK119/K123 ubiquitination in budding as well as fission yeast cells.

Techniques: Expressing, Staining, Quantitation Assay, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: Arabidopsis LSH10 transcription factor interacts with the co-repressor histone deubiquitinase OTLD1 to recruit it to the target promoters

doi: 10.1101/2022.07.30.502139

Figure Lengend Snippet: Association of LSH10 with and effects on ubiquitylation of the chromatin of the OSR2, WUS, ABI5 , and ARL genes. ( A ) Association of LSH10-His6 with the chromatin of the indicated target genes in the lsh10-1/LSH10-His6 plants. A.U., arbitrary units. ( B ) Increase in H2B monoubiquitylation of the OSR2, WUS, ABI5 , and ARL promoter chromatin in the lsh10-1 mutant plants. Wild-type plants, black bars; lsh10-1 , gray bars; lsh10-2 , white bars. Chromatin association of LSH10 and the degree of H2B monoubiquitylation were analyzed by qChIP with primers described in and listed in Table S1. Error bars represent the mean for N>5 biological replicates with 3 technical repeats for each. Numerical values of individual data points are listed in Tables S5 and S6. Differences between mean values assessed by the two-tailed t-test are statistically significant for the p-values * p < 0.05 and ** p < 0.01.

Article Snippet: 5 μl of the crude chromatin extracts were saved for use as an input control, and 100 μl were added into the microwell immobilized with the antibodies (non-specific rabbit IgG Isotype Control (Invitrogen, WB317638) as the negative control, specific rabbit His-tag antibody (GenScript, A00174-40) or anti-monoubiquityl-histone H2B (Lys-120) (5546S, Cell Signaling Technology, Inc.).

Techniques: Mutagenesis, Two Tailed Test