monoclonal antibody against hdac1  (Proteintech)

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Limited proteolysis of endogenous and transiently expressed HDAC1 . Immunoprecipitated endogenous HDAC1 (A) and transiently expressed HDAC1-Flag (HDAC1-F) (B) were incubated at room temperature with increasing concentrations of trypsin (0.000625 μg/μL- 1X, 0.00125 μg/μL- 2X, 0.0025 μg/μL- 4X, 0.005 μg/μL- 8X, 0.01 μg/μL- 16X, and 0.05 μg/μL- 80X). After separation by SDS-PAGE, the proteins were visualized with either anti-HDAC1 antibody in case of endogenous HDAC1 or anti-Flag antibody in case of transiently expressed HDAC1-F. (C) A graph showing percentage of full length endogenous HDAC1 (white) and transiently expressed HDAC1-Flag (black) remaining after exposure to different concentrations of trypsin. The curves were generated by least squares fit to a single exponential. (D) A plot displaying the deacetylase activity of HDAC1-F after incubation with an increasing concentration of trypsin. The inset displays the relationship between deacetylase activity and percentage of full length HDAC1-F remaining after exposure to increasing concentrations of trypsin and the data was fit to a linear equation.

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Immunoprecipitation, Incubation, SDS Page, Generated, Histone Deacetylase Assay, Activity Assay, Concentration Assay

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Predicted trypsin cleavage sites of HDAC1 . The primary sequence of HDAC1 (accession number U50079 ) is shown with arrows indicating the trypsin cleavage sites predicted using the Peptide Cutter program [27]. More information on the sequences, lengths, masses, and cleavage probabilities of the predicted peptide fragments are available [see ].

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Sequencing

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Limited proteolysis of HDAC1 S421A/S423A and HDAC1 E424A/E426A mutants . Immunoprecipitated HDAC1 S421A/S423A (A) and HDAC1 E424A/E426A (B) mutants were incubated with increasing concentrations of trypsin (see Figure 1), separated by SDS-PAGE, and visualized with anti-Flag antibody. The faster migrating proteins present in the absence of trypsin (0X) are derived from the anti-Flag-bound solid phase used for immunoprecipitation [see ]. (C) A graph showing percentage of full length S421A/S423A mutant (red) and E424A/E426A mutant (orange) remaining after exposure to different concentrations of trypsin. The data with wild type HDAC1-F (black- Figure 1) is also included for comparison. The curves were generated by least squares fit to a single exponential.

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Immunoprecipitation, Incubation, SDS Page, Derivative Assay, Mutagenesis, Generated

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Limited proteolysis of HDAC1 S421A and HDAC1 S423A mutants . Immunoprecipitated HDAC1 S421A (A) and HDAC1 S423A (B) mutants were incubated with increasing concentrations of trypsin (see Figure 1), separated by SDS-PAGE, and visualized with anti-Flag antibody. The faster migrating proteins present in the absence of trypsin (0X) are derived from the anti-Flag-bound solid phase used for immunoprecipitation [see ]. (C) A graph showing percentage of full-length HDAC1 S421A (green) and HDAC1 S423A (blue) mutants remaining after exposure to different concentrations of trypsin. The data with wild type HDAC1-F (black- Figure 1) and HDAC1 S421A/S423A (red- Figure 2) proteins are also included for comparison. The curves were generated by least squares fit to a single exponential.

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Immunoprecipitation, Incubation, SDS Page, Derivative Assay, Generated

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Limited proteolysis of HDAC1 E424A and HDAC1 E426A mutants . Immunoprecipitated HDAC1 E424A (A) and HDAC1 E426A (B) mutants were incubated with increasing concentrations of trypsin (see Figure 1), separated by SDS-PAGE, and visualized with anti-Flag antibody. The faster migrating proteins present in the absence of trypsin (0X) are derived from the anti-Flag-bound solid phase used for immunoprecipitation [see ]. (C) A graph showing percentage of full length HDAC1 E424A (green) and HDAC1 E426A (blue) mutants remaining after exposure to different concentrations of trypsin. The data with wild type HDAC1-F (black- Figure 1) and HDAC1 E424A/E426A (orange- Figure 2) proteins are also included for comparison. The curves were generated by least squares fit to a single exponential.

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Immunoprecipitation, Incubation, SDS Page, Derivative Assay, Generated

Journal: BMC Biochemistry

Article Title: Limited proteolysis of human histone deacetylase 1

doi: 10.1186/1471-2091-7-22

Figure Lengend Snippet: Limited proteolysis of HDAC1 H141A mutant . Immunoprecipitated HDAC1 H141A mutant (A) was incubated with increasing concentrations of trypsin (see Figure 1), separated by SDS-PAGE, and visualized with anti-Flag antibody. The faster migrating proteins present in the absence of trypsin (0X) are derived from the anti-Flag-bound solid phase used for immunoprecipitation [see ]. (B) A graph showing percentage of full-length HDAC1 H141A mutant (white) remaining after exposure to different concentrations of trypsin. The data with wild type HDAC1-F (black- Figure 1) are also included for comparison. The curves were generated by least squares fit to a single exponential.

Article Snippet: To immunoprecipitate endogenous HDAC1, the lysate was incubated with 1 μL/mL of monoclonal antibody against HDAC1 (Sigma) for 2 hours prior to addition of 40 μL of protein A agarose beads (Sigma).

Techniques: Mutagenesis, Immunoprecipitation, Incubation, SDS Page, Derivative Assay, Generated

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: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDAC1, HDAC2, and HDAC3 were upregulated in breast cancer tissues and correlated with worse prognosis in breast cancer patients. ( a ) Representative immunohistochemical (IHC) staining photos of HDAC1, HDAC2, and HDAC3 in breast specimens. HDAC1, HDAC2, and HDAC3 levels were obviously elevated in the tumor tissues compared to the non‐neoplastic adjacent tissues of patients with invasive ductal carcinoma (IDC). Yellow boxes indicated mammary ducts. Scale bars, 100 μm. ( b ) Representative IHC photos of three different kinds of HDAC3 subcellular localization. Scale bars, 100 μm. ( c – f ) Overall survival (OS) curves of 139 IDC patients with different HDAC1 ( c ) ( ) HDAC1 Low ( ) HDAC1 High ( ) HDAC1 low‐censored ( ) HDAC1 high‐censored, HDAC2 ( d ) ( ) HDAC2 Low ( ) HDAC2 High ( ) HDAC2 low‐censored ( ) HDAC2 high‐censored, cytoplasmic HDAC3 ( e ) ( ) HDAC3 C‐low ( ) HDAC3 C‐high ( ) HDAC3 C‐low‐censored ( ) HDAC3 C‐high‐censored, and nuclear HDAC3 ( f ) levels ( ) HDAC3 N‐low ( ) HDAC3 N‐high ( ) HDAC3 N‐low‐censored ( ) HDAC3 N‐high‐censored. According to another set of criteria in which cases with either high nuclear or cytoplasmic expression were classified into the C‐high/N‐high group and other cases were classified into the Others group, the overall survival curve of the 139 IDC patients was reproduced as Figure 1g ( ) HDAC3 Others ( ) HDAC3 C‐high/N‐high ( ) HDAC3 Others‐censored ( ) HDAC3 C‐high/N‐high‐censored. P values of the Kaplan‐Meier plots in (c‐g) were calculated by log‐rank test in IBM SPSS Statistics 19 software. ( h – j ) Kaplan‐Meier survival curves with log‐rank analysis were used to assess the correlation between HDAC1 ( h ) ( ) HDAC1 Low (≤ 75th percentile) ( ) HDAC1 High (> 75th percentile), HDAC2 ( i ) ( ) HDAC2 Low (≤ 75th percentile) ( ) HDAC2 High (> 75th percentile), and HDAC3 ( j ) ( ) HDAC3 Low (≤ 80th percentile) ( ) HDAC3 High (> 80th percentile) expression and overall survival of 4903 breast cancer patients in the bc‐GenExMiner platform (website: http://bcgenex.centregauducheau.fr ; all DNA microarray data, node mixed, ER mixed; optimized split for HDAC1 and 2, an 80th percentile customized cutoff for HDAC3).

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Software, Microarray

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in overall survival of IDC patients ( n = 16l)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: Relationship between clinicopathological characteristics and HDACs expression in IDC patients ( n = 139)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Over Expression

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: HDACs expression exhibited different roles in the onset of brain metastasis of IDC patients ( n = 161)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Thoracic Cancer

Article Title: Aberrant HDAC3 expression correlates with brain metastasis in breast cancer patients

doi: 10.1111/1759-7714.13561

Figure Lengend Snippet: The roles of HDACs expression and other clinicopathological characteristics played in the prognosis of breast cancer patients after brain metastasis ( n = 63)

Article Snippet: After serial blocking with hydrogen peroxide and normal horse serum, the tissue chips and sections were incubated with primary monoclonal antibody against HDAC1 (cat. no. 10197‐1‐AP, Proteintech), HDAC2 (cat. no. 12922‐3‐AP, Proteintech) or HDAC3 (cat. no. 10255‐1‐AP, Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Nature Communications

Article Title: Reducing histone acetylation rescues cognitive deficits in a mouse model of Fragile X syndrome

doi: 10.1038/s41467-018-04869-3

Figure Lengend Snippet: FMRP maintains HDAC1 protein levels in NPCs of 6-month-old mice. a , b Western blot analyses of HDAC1 protein levels in WT and Fmr1 KO 6-mo NPCs ( n = 3). c , d HDAC1 protein stability in WT and Fmr1 KO NPCs treated with the protein synthesis inhibitor cycloheximide (CHX) over an 8 h period. Two-way ANOVA ( n = 3, F (4, 20) = 17.86, *** P < 0.001; Time × genotype interaction, F (4, 20) = 2.53, P = 0.0727; Time, F (4, 20) = 2.006, P = 0.1324). Note that FMRP protein levels in WT cells did not change during the treatment. e , f Co-immunoprecipitation analysis showed that endogenous HDAC1 physically associated with endogenous MDM2 in 6-mo NPCs. g Workflow for detection of ubiquitination of HDAC1 using GST-labeled ubiquitin-binding domain (UBD) pull down. h GST-UBD pull down of NPCs with MDM2 knockdown (+shMdm2) and control (−shMdm2) followed by western blotting with a HDAC1 antibody showing that NPCs with reduced MDM2 levels also had reduced ubiquitination of HDAC1. Cells were treated with MG132 to inhibit proteasome degradation of ubiquitinated proteins. i Schematic models of FMRP balancing histone acetylation through regulation of MDM2-HDAC1 and EP300 pathways. ** P < 0.01, *** P < 0.001, except for Fig. 3d, Student’s t -tests were used for data analyses. Data are presented as mean ± s.e.m.

Article Snippet: A monoclonal antibody against HDAC1 (BioVersion, 3601-30) or MDM2 (Santa Cruz, sc-965) was incubated with AminoLink Plus coupling Resin for 2 h and washed 3 times with coupling buffer (10 mM sodium phosphate, 150 Mm NaCl; pH 7.2).

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Labeling, Binding Assay, Knockdown, Control

Journal: Nature Communications

Article Title: Reducing histone acetylation rescues cognitive deficits in a mouse model of Fragile X syndrome

doi: 10.1038/s41467-018-04869-3

Figure Lengend Snippet: Increasing HDAC1 rebalances histone acetylation and rescues proliferation and differentiation deficits of FMRP-deficient NPCs. a A schematic model showing that enhancing HDAC1 activities through either pharmacological or genetic reduction of MDM2 rebalances histone acetylation and rescues proliferation and differentiation of Fmr1 KO NPCs. b , c Western blot analyses of HDAC1 and FMRP protein levels in 6-mo NPCs treated with Nutlin-3 ( n = 3). GAPDH was used as a loading control. d – f Western blot analyses of total and acetylated histone H3 ( d , e ) and histone H4 ( d , f ) in NPCs treated with Nutlin-3 ( n = 3). Total histone H3 and total histone H4 were used as loading controls for acetylated H3 and H4, respectively. g , h Nutlin-3 treatment rescued the cell proliferation phenotype of Fmr1 KO NPCs as measured by BrdU incorporation ( g , red; scale bar, 20 µm) followed by quantitative analysis ( h , n = 3). i , j Nutlin-3 treatment rescued neuronal differentiation phenotypes of Fmr1 KO NPCs as assessed by Tuj1 + staining ( i , red; scale bar, 20 µm) followed by quantitative analysis ( j , n = 3). k , l Acute knockdown of MDM2 rescued the cell proliferation phenotype of Fmr1 KO NPCs as assessed by incorporation of the thymidine analog, BrdU ( l , red, BrdU; green, GFP, scale bar, 20 µm) and quantitative analysis ( l , n = 3). m , n Acute knockdown of EP300 rescued neuronal differentiation phenotypes of Fmr1 KO NPCs as assessed by a neuronal marker Tuj1 + ( m , red, Tuj1; green, GFP, scale bar, 20 µm) followed by quantitative analysis ( n , n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: no significant difference. Two-way ANOVA was used for all data analyses. Data are presented as mean ± s.e.m.

Article Snippet: A monoclonal antibody against HDAC1 (BioVersion, 3601-30) or MDM2 (Santa Cruz, sc-965) was incubated with AminoLink Plus coupling Resin for 2 h and washed 3 times with coupling buffer (10 mM sodium phosphate, 150 Mm NaCl; pH 7.2).

Techniques: Western Blot, Control, BrdU Incorporation Assay, Staining, Knockdown, Marker

Journal: Nature Communications

Article Title: Reducing histone acetylation rescues cognitive deficits in a mouse model of Fragile X syndrome

doi: 10.1038/s41467-018-04869-3

Figure Lengend Snippet: Models for FMRP maintain histone acetylation through regulating both EP300 and HDAC1 expression levels, which impact adult neurogenesis and cognition

Article Snippet: A monoclonal antibody against HDAC1 (BioVersion, 3601-30) or MDM2 (Santa Cruz, sc-965) was incubated with AminoLink Plus coupling Resin for 2 h and washed 3 times with coupling buffer (10 mM sodium phosphate, 150 Mm NaCl; pH 7.2).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: RBM45 competes with HDAC1 for binding to FUS in response to DNA damage

doi: 10.1093/nar/gkx1102

Figure Lengend Snippet: RBM45 competes with HDAC1 for binding with FUS. ( A ) RBM45 interacts with HDAC1. After transfected with the indicated plasmids, cells were lysed and immunoprecipitated by antibodies against HA or IgG, and the bound proteins were analyzed by immunoblotting with anti-Flag monoclonal antibody. ( B ) Depletion of FUS decreases the interaction between RBM45 and HDAC1. 48 h after expressing siNC or siFUS, 293T cells were cotransfected with Flag-RBM45 and HA-HDAC1, then lysed and immunoprecipitated by anti-HA antibody. The bound proteins were analyzed by immunoblotting with anti-Flag. ( C ) RBM45 knockdown enhances the cooperation between FUS and HDAC1. 293T cells were transfected with siNC or siRBM45 before co-transfected with flag-FUS and HA-HDAC1. Anti-HA and anti-Flag antibodies were used for precipitation and blotting, respectively. ( D ) HDAC1 knockdown increases the interaction between RBM45 and FUS. 293T cells expressing siRNA were co-transfected with flag-FUS and HA-RBM45. Anti-Flag M2 beads were used for immunoprecipitation, and anti-HA antibody was used for immunoblotting.

Article Snippet: Antibodies used in this study included rabbit polyclonal antibodies against RBM45 (Abcam, Ab123912), HA-tag (Biolegend, 902302), Flag (Sigma, 7425), H3.1 (Abmart, p30266); mouse monoclonal antibodies against HDAC1 (Abcam, Ab31263), phosphor-H2AX (Millipore, 05-636-AF555), FUS (Santa Cruz, sc-4771), PARP1 (Santa Cruz, sc8007), β-actin (Protein tech, 00001-1), Biotin (Terminal)-PAR (Trevigen, 4336-100-02).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: RBM45 competes with HDAC1 for binding to FUS in response to DNA damage

doi: 10.1093/nar/gkx1102

Figure Lengend Snippet: RBM45 regulates the recruitment of HDAC1. ( A and B ) Depletion of RBM45 increases the percentage of cells with HDAC1 accumulation. U2OS cells were transfected with siNC or siRBM45. 48 h later, cells were laser micro-irradiated, fixed and stained with HDAC1 and γH2AX antibodies. Cells with HDAC1 recruited to the laser-induced sites of damage were counted. Data are presented as mean ± SD. n = 2; 30–40 irradiated cells per experiment. P values from an unpaired t test were included. ( C ) RBM45 knockdown enhances the enrichment of HDAC1 on chromatin. Chromatin fractions from siNC- or siRBM45-treated HeLa cells were extracted at 30 min after X-ray irradiation followed by immunoblotting with anti-HDAC1 antibody. ( D ) Overexpression of RBM45 inhibits the binding of HDAC1 to chromatin. Chromatin fractions from HeLa cells expressing HA-vector or HA-RBM45 were extracted followed by western blotting with anti-HDAC1 antibody. ( E ) Depletion of RBM45 affects H4K16ac. 72 h after transfected with siNC or siRBM45, cells were irradiated with X-ray (10 Gy). 0 or 30 min later, cells were harvested for immunoblotting. ( F ) Overexpression of RBM45 affects H4K16ac. 24 h after transfected with either HA or HA-RBM45, cells were treated as in (E). ( G, H ) RBM45 affects the chromatin structure. HeLa cells expressing siNC or siRBM45 were digested with micrococcal nuclease (MNase, MN) for 4 or 8 min. The genomic DNA was analyzed using agarose gel electrophoresis (G). The position of size markers were shown at the left. The intensities of lane 2, 5, 8 were consecutively quantified by using Quantity One software (Bio-Rad). N2–N4 reflect the number of nucleosomes in each oligonucleosome (H). The asterisk is a nonspecific signal.

Article Snippet: Antibodies used in this study included rabbit polyclonal antibodies against RBM45 (Abcam, Ab123912), HA-tag (Biolegend, 902302), Flag (Sigma, 7425), H3.1 (Abmart, p30266); mouse monoclonal antibodies against HDAC1 (Abcam, Ab31263), phosphor-H2AX (Millipore, 05-636-AF555), FUS (Santa Cruz, sc-4771), PARP1 (Santa Cruz, sc8007), β-actin (Protein tech, 00001-1), Biotin (Terminal)-PAR (Trevigen, 4336-100-02).

Techniques: Transfection, Irradiation, Staining, Western Blot, Over Expression, Binding Assay, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Software

Journal: Nucleic Acids Research

Article Title: RBM45 competes with HDAC1 for binding to FUS in response to DNA damage

doi: 10.1093/nar/gkx1102

Figure Lengend Snippet: FUS mutant R521C prefers to interact with RBM45 than HDAC1. (A, B) FUS- R521C mutation in fALS enhances its interaction with RBM45. ( A ) Cells co-transfected with Flag-FUS or Flag-FUS-R521C with HA-RBM45 were harvested for immunoprecipitation using M2 beads. Antibodies against HA or Flag were used for blotting. ( B ) Purified GST-FUS or GST-FUS-R521C was incubated with cell lysate expressing HA-RBM45. Antibody against HA was used for blotting. Asterisks mean specific bands. ( C ) FUS-R521C mutation promotes RBM45 recruitment to the damage sites. 48 h after expressing siNC or siFUS, cells were transfected with Flag-FUS or Flag-FUS-R521C as well as GFP-RBM45, followed by microirradiation. Data are presented as mean ± SD. n = 2; 30–40 irradiated cells per experiment. P values from unpaired t test were included. ( D ) FUS-R521C mutation does not affect its interaction with HDAC1 in vitro . Purified GST-FUS or GST-FUS-R521C was incubated with cell lysates expressing HA-HDAC1. Antibody against HA was used for blotting. Asterisks mean specific bands. ( E ) Depletion of RBM45 stimulates the association between FUS-R521C and HDAC1 in vivo . 293T cells transfected with siNC or siRBM45 were co-transfected with Flag-FUS-R521C and HA-HDAC1. Anti-HA was used for immunoprecipitation. ( F ) RBM45 knockdown makes FUS-R521C functional in promoting HDAC1 recruitment. 48 h after transfection of siRNAs targeting FUS and RBM45, cells were transfected with FUS or FUS-R521C followed by microirradiation. Cells were then stained with antibody against HDAC1. Data are presented as mean ± SD. n = 2; 30–40 irradiated cells per experiment.

Article Snippet: Antibodies used in this study included rabbit polyclonal antibodies against RBM45 (Abcam, Ab123912), HA-tag (Biolegend, 902302), Flag (Sigma, 7425), H3.1 (Abmart, p30266); mouse monoclonal antibodies against HDAC1 (Abcam, Ab31263), phosphor-H2AX (Millipore, 05-636-AF555), FUS (Santa Cruz, sc-4771), PARP1 (Santa Cruz, sc8007), β-actin (Protein tech, 00001-1), Biotin (Terminal)-PAR (Trevigen, 4336-100-02).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Purification, Incubation, Expressing, Irradiation, In Vitro, In Vivo, Functional Assay, Staining

Journal: Nucleic Acids Research

Article Title: RBM45 competes with HDAC1 for binding to FUS in response to DNA damage

doi: 10.1093/nar/gkx1102

Figure Lengend Snippet: Models of RBM45 in DNA damage response under normal and ALS mutation conditions. ( A ) RBM45 relocalizes to the sites of DNA damage in a way dependent on PAR and FUS to prevent excessive recruitment of HDAC1. ( B ) FUS-R521C, a mutant in fALS patients, enhances its interaction with RBM45, which decreases its association with HDAC1 and diminishes the recruitment of HDAC1.

Article Snippet: Antibodies used in this study included rabbit polyclonal antibodies against RBM45 (Abcam, Ab123912), HA-tag (Biolegend, 902302), Flag (Sigma, 7425), H3.1 (Abmart, p30266); mouse monoclonal antibodies against HDAC1 (Abcam, Ab31263), phosphor-H2AX (Millipore, 05-636-AF555), FUS (Santa Cruz, sc-4771), PARP1 (Santa Cruz, sc8007), β-actin (Protein tech, 00001-1), Biotin (Terminal)-PAR (Trevigen, 4336-100-02).

Techniques: Mutagenesis