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: Acetylated proteins more than two-fold up regulated by nutlin-3 detected by SILAC-based mass spectrometry

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: Sequencing

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: Nutlin-induced modulation of acetylated proteins in the AML cell line MOLM-13. (A) MOLM-13 cells were subjected to stable isotope labeling with amino acids in cell culture (SILAC). Cells were labeled with either light (L-Lysine-2HCl, L-Arginine-HCl) or heavy ( 13 C 6 L-Lysine-2HCl, 13 C 6 15 N 4 L-Arginine-HCl) isotopes of amino acids and treated with DMSO (control) or 6 μM nutlin-3, respectively, for 6 hours. Cells were harvested and lysed, and lysates were mixed at a ratio of 1:1 (5 mg protein of each). The lysate was precleared with uMACs protein G Microbeads, then precleared with beads and an unspecific antibody (rabbit IgG), before immunoprecipitation of acetylated proteins using an anti-acetyl-lysine antibody and Microbeads. Proteins were eluted in 95°C SDS loading buffer and subjected to one-dimensional gel electrophoresis (SDS-PAGE) and staining with Coomassie Blue. Bands were excised and peptides generated by trypsination. Peptides were separated and fragmented using LC-MS/MS (LC-LTQ-Orbitrap) and protein ID’s and H/L ratios were obtained using MaxQuant and Perseus software. (B) Representative MS/MS spectrum of peptides derived from Histone H2B.

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: Labeling, Cell Culture, Immunoprecipitation, Nucleic Acid Electrophoresis, SDS Page, Staining, Generated, Liquid Chromatography with Mass Spectroscopy, Software, Tandem Mass Spectroscopy, Derivative Assay

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: PLoS ONE

Article Title: Identification of Protein Network Alterations upon Retinal Ischemia-Reperfusion Injury by Quantitative Proteomics Using a Rattus norvegicus Model

doi: 10.1371/journal.pone.0116453

Figure Lengend Snippet: The representative western blots of β-actin, albumin, calretinin, SYPH, SYT-1, Annexin A1, ApoA4, calnexin, GNAL, GFAP, H2B, HSP90AB1 and vimentin shown as the expression levels in the retinas (A) and as the densitometric quantitative results (B). Equal amounts of protein from the control retinas and the I/R-treated retinas were loaded. Each lane represents individual retina (n = 4 or 6 in each group; *p<0.05 compared with the non-injured retinas). The error bars represent the standard error of the mean. C, Retinal I/R-induced reductions of the synaptic proteins SYPH and SYT-1. The representative western blots of synaptophysin (SYPH) and synaptotagmin-1 (SYT-1) shown as the expression levels in the retinas are seen in (A). The stained retinal sections of SYPH (B) and SYT-1 (C) are shown at 2 days after the injury. Blue color: DAPI stained nuclei as control. Red color: SYPH or SYT-1 positively stained synapses. The scale bar represents 50 µm. C, non-injured eyes; I/R, I/R-injured eyes.

Article Snippet: Five microgram of the proteins from in vivo and in vitro samples were fractionated by SDS-PAGE, electroblotted onto a PVDF membrane (Millipore, Billerica, MA) and probed with different antibodies: anti-Calretinin (1∶5000 dilution, #2449-1, Abcam, CA), anti-actin (1∶2000 dilution, #CW0096B, CWBIO, China), anti-Albumin (1∶10000 dilution, 16475-1-AP, Proteintech, China), anti-synaptophysin (SYPH) (1∶50000 dilution, #1485-1, Abcam, CA), anti-SYT1 (1∶10000 dilution, 14511-1-AP, Proteintech, China), anti-mTOR (1∶1000 dilution, #2983, CST, MA), anti-phospho-mTOR (Ser2448) (1∶1000 dilution, #2971, CST, MA), anti-phospho-p70 S6 Kinase (Ser371) (1∶1000 dilution, #9208, CST, MA), anti-4E-BP1 (1∶1000 dilution, #9644, CST, MA), anti-phospho-4E-BP1 (Thr37/46) (1∶1000 dilution, #2855, CST, MA), anti-GFAP (1∶5000 dilution, #7260, Abcam, CA), anti-vimentin (1∶10000 dilution, #92560, Millipore, MA), anti-ApoA4 (1∶3000 dilution, #5700, CST, MA), anti-GNAL (1∶2000 dilution, #DF4108, Affinity, OH), anti-HSP90AB1 (1∶1000 dilution, #A1087, ABclonal, MA), anti-H2B (1∶1000 dilution, #8135, CST, MA), anti-Annexin A1 (1∶2000 dilution, 21990-1-AP, Proteintech, China) and anti-calnexin (1∶5000 dilution, #2692-1, Abcam, CA).

Techniques: Western Blot, Expressing, Staining

Journal: eLife

Article Title: High-intensity interval training remodels the proteome and acetylome of human skeletal muscle

doi: 10.7554/eLife.69802

Figure Lengend Snippet: HIIT increased acetylation on ( A ). H1.5 K48, ( B ). H2B type 2F K16/20 and ( C ). H3 K23. ( D ). HIIT did not alter H4 acetylation (n=7). ( E ) Immunoblotting analysis identified the upregulation of the nuclear-localized acetyltransferase EP300 (n=8, mean of three technical replicates; participant 1 was excluded as an outlier from statistical analysis (pre, post and fold-change values were all >3 median absolute deviations from the respective median), data for participant 1 is shown in the translucent data points). ( F ) Knockdown of EP300 reduces acetylation of H2B K20 but not H3 K23 in C2C12 myotubes (n=3). Representative images confirming equal loading are displayed in . Summary statistics are mean ± SEM. † Π<0.05. * p<0.05, ** p<0.01, *** p<0.001. Figure 7—source data 1. Full image and annotation of H2B immunoblot. Figure 7—source data 2. Raw ImageLab file of H2B immunoblot. Figure 7—source data 3. Full image and annotation of H2B K20 immunoblot. Figure 7—source data 4. Raw ImageLab file of H2B K20 immunoblot. Figure 7—source data 5. Raw quantification data of H2B K20/H2B immunoblots. Figure 7—source data 6. Full image and annotation of H3 immunoblot. Figure 7—source data 7. Raw ImageLab file of H3 immunoblot. Figure 7—source data 8. Full image and annotation of H3 K23 immunoblot. Figure 7—source data 9. Raw ImageLab file of H3 K23 immunoblot. Figure 7—source data 10. Raw quantification data of H3 K20/H3 immunoblots. Figure 7—source data 11. Full image and annotation of EP300 immunoblots (human skeletal muscle). Figure 7—source data 12. Raw ImageLab file of EP300 immunoblots (human skeletal muscle, replicate 1). Figure 7—source data 13. Raw ImageLab file of EP300 immunoblots (human skeletal muscle, replicates 2 & 3). Figure 7—source data 14. Raw quantification data of EP300 immunoblot (human skeletal muscle).

Article Snippet: Antibody , H2B Antibody, rabbit monoclonal , Cell Signaling Technologies , Cat#12364 RRID: AB_2714167 , (1:3000).

Techniques: Western Blot

Journal: eLife

Article Title: High-intensity interval training remodels the proteome and acetylome of human skeletal muscle

doi: 10.7554/eLife.69802

Figure Lengend Snippet:

Article Snippet: Antibody , H2B Antibody, rabbit monoclonal , Cell Signaling Technologies , Cat#12364 RRID: AB_2714167 , (1:3000).

Techniques: Electrophoresis, Western Blot, Bicinchoninic Acid Protein Assay, Sequencing, Software, Lysis, Protein Concentration, Imaging, Staining, Chromatography, Sample Prep

Journal: Molecular cell

Article Title: Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

doi: 10.1016/j.molcel.2021.06.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Histone H2B (V119) , Cell Signaling Technology , Cat# 8135; RRID: AB_10891053.

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Imaging, CCK-8 Assay, CRISPR, shRNA, Negative Control, Construct, Software, Transfection, DNA Extraction