mouse antibodies against hdac1  (Cell Signaling Technology Inc)

Journal: Leukemia

Article Title: Ibrutinib downregulates a subset of miRNA leading to upregulation of tumor suppressors and inhibition of cell proliferation in chronic lymphocytic leukemia

doi: 10.1038/leu.2016.181

Figure Lengend Snippet: mRNA transcripts of 43 predicted targets of miR-22, miR-34a, miR-146b, and miR-181b were counted by nanoString nCounter assay with RNA isolated from 17 paired pre- and on ibrutinib treatment (day 28) CLL cells. Nine out of 43 predicted targets were significantly altered on ibrutinib (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN, and SMAD4). Scatter plot graph showing the fold change post-ibrutinib:pre-ibrutinib expression ratio, for individual patient CLL cells for nine miRNA targets. A line at 0 is inserted to separate positive and negative fold changes. Median and interquartile ranges are represented. P values were calculated by Wilcoxon paired test. *, p < 0.05, **, p < 0.01.

Article Snippet: Mouse monoclonal antibody against HDAC1 (#05–100-I) was purchased from EMD Millipore (Billerica, MA, USA).

Techniques: Isolation, Expressing

Journal: Leukemia

Article Title: Ibrutinib downregulates a subset of miRNA leading to upregulation of tumor suppressors and inhibition of cell proliferation in chronic lymphocytic leukemia

doi: 10.1038/leu.2016.181

Figure Lengend Snippet: The scrambled control, pre-miR-34a and pre-miR-146b oligos were transfected into SP53 and MEC1 cells, respectively. The expression of miRNAs and putative tumor suppressor mRNAs was measured by qPCR in triplicate. Mean and standard deviation are shown. (a) The expression of miR-34a and miR-146b were markedly increased compared with the scramble control transfection in both SP53 and MEC1 cells 30 hours after pre-miR-34a and pre-miR-146b transfection. (b) The expression of the putative tumor suppressor mRNAs was significantly reduced in SP53 and MEC1 cells after pre-miR-34a and pre-miR-146b transfection. For (a) and (b), P values were calculated by the Student’s t-test. *, p < 0.05, **, p < 0.01; ***, p < 0.001; ● , p =0.06. (c) and (d) Western blot and densitometry analysis data showing decreased protein expression of ATM, SMAD4, and HDAC1 in SP53 cells transfected with pre-miR-34a (c) and pre-miR-146b (d), in comparison to scramble control.

Article Snippet: Mouse monoclonal antibody against HDAC1 (#05–100-I) was purchased from EMD Millipore (Billerica, MA, USA).

Techniques: Control, Transfection, Expressing, Standard Deviation, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Nucleoprotein (NP) interacted with histone deacetylase 1 (HDAC1) in vivo and in vitro . (A) Myc-NP was co-expressed with HA-tagged HDAC1, HDAC2, HDAC3, or HDAC8 in HEK293T cells, and immunoprecipitates by anti-Myc antibody. The samples were blotted with indicated antibodies. GAPDH was used as a loading control. (B) 293T cells were infected with H5N1, H1N1, or H7N9 virus at an MOI of 1. 24 h postinfection, the cell lysates were subjected to immunoprecipitation with control IgG or NP antibodies and subsequently immunoblotted with indicated antibodies. (C) A549 cells were infected with H5N1, H1N1, or H7N9 virus at an MOI of 1. 12 h postinfection, cells were fixed and stained by NP antibody (green) and HDAC1 (red). The Pearson’s correlation (r) of NP-HDAC1 co-localization was calculated by Imaris, at least 40 cells in each group were scored. The nucleus was stained with DAPI (blue). Scale bar: 5 µm. Data are representative of three independent experiments. (D) 6His-NP (20 µg) immobilized on nickel-nitrilotriacetic acid (Ni-NTA) beads were incubated with/without recombinant HDAC1 (20 µg), the beads were then extensively washed, and the proteins resolved by SDS-PAGE and detected the NP and HDAC1 by Western blot. (E) In vitro deacetylation assay. 293T-expressed NP-6His proteins (5 µg) were purified and washed with high salt buffer to remove the binding partners, then incubated with glutathione S-transferase (GST)-HDAC1 (1 µg) in HDAC buffer for 60 min at 30°C. The proteins were resolved by SDS-PAGE and detected the Ace-NP, NP, and HDAC1 by Western blot.

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Histone Deacetylase Assay, In Vivo, In Vitro, Control, Infection, Virus, Immunoprecipitation, Staining, Incubation, Recombinant, SDS Page, Western Blot, Purification, Binding Assay

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Nucleoprotein (NP) is acetylated on the lysine residue at amino acid position 103. (A) HEK293T cells were transfected with the NP with a Myc-tagged at the C-terminal. The Myc-NP (red arrow indicated) band was analyzed by mass spectrometry. (B) The protein was identified as the influenza A viruses (IAV) [A/environment/Qinghai/1/2008(H5N1)] NP. Blue indicates peptides detected by mass spectrometry; red indicates the acetylated lysine sites. (C) K103 is the primary acetyl amino acid suggested in mass spectrometry. (D) The expression of lysine to alanine substitution mutants of NP. HEK293T cells were transfected with NP-Myc WT or mutants. WT and mutant NP were purified by Myc-tag pull-down followed by Western blotting (IB) with the anti-myc antibody (Myc) or anti-acetylated lysine antibody (Ace-Lys). β-actin was used as a loading control. (E) The relative expression level of Ace-NP protein in (D) was quantified by imageJ. (F) Histone deacetylase 1 (HDAC1) deacylated the acylation of NP at K103. HEK293T cells expressing indicated proteins were subjected to immunoprecipitation using anti-Myc antibody, the samples were immunoblotted with the indicated antibodies. β-actin was used as a loading control. (G) The relative Ace-NP protein expression level in (F) was quantified by using imageJ. Statistical significance was determined by Student’s t- test. * P < 0.5 versus WT group. Data are representative of three independent experiments [mean ± SD in (E,G) ].

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Residue, Transfection, Mass Spectrometry, Expressing, Mutagenesis, Purification, Western Blot, Control, Histone Deacetylase Assay, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Histone deacetylase 1 (HDAC1) regulated the localization of nucleoprotein (NP) independent the K103 acylation. A549 cells were transfected with siCONT or siHDAC1 for 48 h and then infected with rWT, rK103A, or rK103R viruses at MOI of 1. (A) At 24 h postinfection, the subcellular distribution of the NP (green) and HDAC1 (red) was analyzed by IFAs. The nucleus was stained with DAPI (blue). (B) More than 400 cells in each group were scored. Nuclear and cytoplasmic (N + C), predominantly nuclear (N), predominantly cytoplasmic (C). * P < 0.05; ** P < 0.01 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments [mean ± SD in (B) ]. Scale bar: 10 µm. (C) HEK293T cells were transfected siCONT or siHDAC1 for 48 h and then co-transfected with pPolI–Luc, viral protein polymerase basic protein 2 (PB2), PB1, polymerase acidic protein (PA) expression plasmids, with or without NP (WT or mutants). Renilla luciferase was used as an internal control.

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Histone Deacetylase Assay, Transfection, Infection, Staining, Expressing, Luciferase, Control

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Effects of histone deacetylase 1 (HDAC1) on influenza A viruses (IAV) infection. (A) Knockdown of HDAC1 expression was shown by immunoblot analysis, using HDAC1-specific antibody. Four separate short hairpin HDAC1 (shHDAC1) lentivirus clones were used. shCONT was used as the lentivirus infection control. β-actin was used as a loading control. (B–E) Depletion of HDAC1 decreased viral nucleoprotein (NP) protein level and inhibited IAV infection. A549 cells with/without shHDAC1 as indicated were infected with NP rWT and mutant virus at an MOI of 1 for 24 h, the virion from the culture medium was collected and subjected to Western blot (B) and plaque assay on MDCK cells (C) . A549 cells with/without siHDAC1 as indicated were infected with rWT, rK103A, or rK103R virus at an MOI of 1, the virion from the culture medium was collected and subjected to plaque assay on MDCK cells (D) and Western blot (E) . (F–H) Effect of HDAC1 overexpression on IAV infection. A549 cells were transfected with/without pcDNA3.0-HDAC1. 36 h posttransfection, cells were infected with rWT/rK103R virus at an MOI of 1 for 24 h. Total lysate of the infected cells was collected for Western blot (F) . The virion from the culture medium was collected and subjected to plaque assay on MDCK cells (G) and Western blot (H) . * P < 0.05; ** P < 0.01, *** P < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments [mean ± SD in (C,D,G) ].

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Histone Deacetylase Assay, Infection, Knockdown, Expressing, Western Blot, Clone Assay, Control, Mutagenesis, Virus, Plaque Assay, Over Expression, Transfection

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Depletion of histone deacetylase 1 (HDAC1) inhibited influenza A viruses (IAV) infection through activation the TBK1-IRF3 and ERK signaling pathways. A549 knockdown HDAC1 stable cells and control cells (shCONT) were infected with rWT/rK103A/rK103R virus at an MOI of 1 for 24 h. (A–E) The relative expression level of nucleoprotein (NP) (A) , HDAC1 (B) , IFNα (C) , IL6 (D) , and TLR3 (E) were determined by qRT-PCR. (F) Total lysate of the infected cells was subjected for Western blot analysis of viral NP, Ace-NP, HDAC1, p-IRF3, IRF3, p-TBK1, TBK1, p-ERK, and ERK. β-actin was used as a loading control. (G) The relative Ace-NP expression level equal to the Ace-NP level divided by the total NP. * P < 0.05; ** P < 0.01, *** P < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments [mean ± SD in (A–E) ].

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Histone Deacetylase Assay, Infection, Activation Assay, Protein-Protein interactions, Knockdown, Control, Virus, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

doi: 10.3389/fimmu.2017.01757

Figure Lengend Snippet: Histone deacetylase 1 (HDAC1) was a possible switch for regulation of the TBK1-IRF3 signaling pathways. (A) A549 cells were infected with H5N1 virus at an MOI of 0.5 and collected at indicated time postinfection. (B) Nucleoprotein (NP), HDAC1, and IFN-α levels were quantified relative to GAPDH and by densitometry using ImageJ. (C,D) HEK293T cells were transfected with gradient amount of NP for 36 h, protein (C) , and mRNA (D) level of HDAC1 and NP were analyzed. Data are representative of three independent experiments [mean ± SD in (B,D) ]. (E) A possible schematic model: K103 of NP was an acetylation site regulated by HDAC1. Two K103 NP mutants, K103A and K103R, lacked of regulation by HDAC1, enhanced NP nuclear localization and replication efficiency of the recombinant viruses in vitro . Depletion of HDAC1 inhibited influenza A virus replication via Two paths: one was decreased the RNP activity, one was activated TBK1-IRF3 signaling.

Article Snippet: Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Histone Deacetylase Assay, Protein-Protein interactions, Infection, Virus, Transfection, Recombinant, In Vitro, Activity Assay