PA1-860

antibody from Invitrogen Antibodies

Targeting: HDAC1 GON-10, HD1, KDAC1, RPD3L1

Provider product page for PA1-860

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [68]
Comments [0]
Validations
Western blot [7]
Immunocytochemistry [5]
Immunohistochemistry [3]
Chromatin Immunoprecipitation [1]
Other assay [39]

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Product number: PA1-860 - Provider product page
Provider: Invitrogen Antibodies
Product name: HDAC1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-860 detects histone deacetylase 1 (HDAC1) from human, mouse, rat, hamster and canine tissues and cells. PA1-860 has been successfully used in Western blot, immunoprecipitation, ChIP, immunofluoresence, immunocytochemistry and immunohistochemistry procedures. By Western blot, this antibody detects an ~62 kDa protein representing HDAC1 from HeLa cell lysate. Immunocytochemical staining of HDAC1 in HeLa cells with PA1-860 results in nuclear staining. The PA1-860 immunogen is a synthetic peptide corresponding to residues C E(467) E K P E A K G V K E E V K L A(482) of human HDAC1. This immunizing peptide (Cat. # PEP-069) is available for use in neutralization and control experiments.
Reactivity: Human, Mouse, Rat, Canine, Hamster
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

HDAC1 protein structure - PA1-860 shown in red.

Supportive validation

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Experimental details: Western blot analysis of HDAC1. 50 µg total protein lysate from human embryonic kidney HEK293T cells (lane 2) and mouse myoblast C2C12 cells (lane 3) in 1X NuPAGE® LDS Sample Buffer (Product # NP0007) + 1mM DTT loaded onto a NuPAGE™ Novex™ 4-12% Bis-Tris protein gel (Product # NP0321PK2). Molecular weight marker loaded for size reference (lane 1). Gel run in 1X NuPAGE® MOPS SDS Running Buffer (Product # NP0001) at room temperature and then transferred to a methanol-activated Novex™ PVDF membrane (Product # LC2002) using a Invitrogen Mini Blot Module (Product # B1000) in 1X NuPAGE® Transfer Buffer (Product # NP0006) at 20V for 2 hours at room temperature. Membrane was rinsed in 1X TBS-T and incubated in blocking solution (5% milk in 1X TBS-T) for 1 hour at room temperature on a rocking platform. HDAC1 detection was performed by incubating blot with rabbit polyclonal anti-HDAC1 antibody (Product # PA1-860; 1 mg/mL) diluted at 1:1000 in blocking solution for 1 hour at room temperature on a rocking platform. Primary antibody was washed 10 times with 1X TBS-T over the course of 1 hour at room temperature on a rocking platform. Secondary antibody binding was performed by incubation with goat anti-rabbit IgG-HRP secondary diluted at 1:5000 in blocking solution for 1 hour at room temperature on a rocking platform. Following washes (as above), chemiluminescent imaging was performed. Data courtesy of Dr. Raj Jain's laboratory.

Supportive validation

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Experimental details: Western blot analysis of HDAC1. 30 µg total protein lysate from MEFs (lane 2) and HEK293T cells (lane 3) in 1X NuPAGE® LDS Sample Buffer (Product # NP0007) + 1X NuPAGE® Sample Reducing Agent (Product # NP0009) loaded onto a NuPAGE™ Novex™ 4-12% Bis-Tris protein gel (Product # NP0321PK2). Gel run in 1X NuPAGE® MOPS SDS Running Buffer (Product # NP0001) at room temperature and then transferred to a methanol-activated Novex™ PVDF membrane (Product # LC2002) using a Invitrogen Mini Blot Module (Product # B1000) in 1X NuPAGE® Transfer Buffer (Product # NP0006) at 20V for 2 hours at room temperature. Membrane was washed for 1 minute in 1X TBS-T and incubated in blocking solution (5% milk in 1X TBS-T) for 1 hour at room temperature on a rocking platform. HDAC1 detection was performed by incubating blot with rabbit polyclonal anti-HDAC1 antibody (Product # PA1-860) diluted at 1:1000 in blocking solution for 1 hour at room temperature on a rocking platform. Primary antibody was washed 10 times with 1X TBS-T over the course of 1 hour at room temperature on a rocking platform. Secondary antibody binding was performed by incubation with goat anti-rabbit IgG-HRP diluted at 1:5000 in blocking solution for 1 hour at room temperature on a rocking platform. Following washes (as above), chemiluminescent detection was performed. Data courtesy of Antibody Data Exchange Program.

Supportive validation

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Experimental details: Western blot of HDAC1 on HeLa cell extract using Product # PA1-860.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HDAC1. 50 µg total protein lysate from human embryonic kidney HEK293T cells (lane 2) and mouse myoblast C2C12 cells (lane 3) in 1X NuPAGE® LDS Sample Buffer (Product # NP0007) + 1mM DTT loaded onto a NuPAGE™ Novex™ 4-12% Bis-Tris protein gel (Product # NP0321PK2). Molecular weight marker loaded for size reference (lane 1). Gel run in 1X NuPAGE® MOPS SDS Running Buffer (Product # NP0001) at room temperature and then transferred to a methanol-activated Novex™ PVDF membrane (Product # LC2002) using a Invitrogen Mini Blot Module (Product # B1000) in 1X NuPAGE® Transfer Buffer (Product # NP0006) at 20V for 2 hours at room temperature. Membrane was rinsed in 1X TBS-T and incubated in blocking solution (5% milk in 1X TBS-T) for 1 hour at room temperature on a rocking platform. HDAC1 detection was performed by incubating blot with rabbit polyclonal anti-HDAC1 antibody (Product # PA1-860; 1 mg/mL) diluted at 1:1000 in blocking solution for 1 hour at room temperature on a rocking platform. Primary antibody was washed 10 times with 1X TBS-T over the course of 1 hour at room temperature on a rocking platform. Secondary antibody binding was performed by incubation with goat anti-rabbit IgG-HRP secondary diluted at 1:5000 in blocking solution for 1 hour at room temperature on a rocking platform. Following washes (as above), chemiluminescent imaging was performed. Data courtesy of Dr. Raj Jain's laboratory.

Supportive validation

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Experimental details: Knockout of HDAC1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1087187_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HDAC1 was performed by loading 30 µg of HeLa Wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa HDAC1 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HDAC1 Polyclonal Antibody (Product # PA1-860, 0.1 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to HDAC1. Uncharacterized band was observed in all the samples at 45 kDa.

Supportive validation

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Experimental details: Knockdown of HDAC1 was achieved by transfecting MCF7 cells with HDAC1 specific siRNAs (Silencer® select Product # s73). Western blot analysis (Fig. a) was performed using Modified whole cell extracts (1% SDS) from the HDAC1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-HDAC1 Polyclonal Antibody (Product # PA1-860, 1µg/ml) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to HDAC1..

Supportive validation

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Experimental details: Western blot was performed using Anti-HDAC1 Polyclonal Antibody (Product # PA1-860) and ~55kDa band corresponding to HDAC1 was observed in MCF-7, HeLa, HEK-293, NIH/3T3 and PC-12 cells. Modified whole cell extracts (1% SDS) (30 ug lysate) of MCF-7 (Lane 1), HeLa (Lane 2), HEK-293 (Lane 3), NIH/3T3 (Lane 4) and PC-12 (Lane 5) were electrophoresed using NuPAGE® 4-12% Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001).The blot was probed with the primary antibody (1µg/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

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Experimental details: Immunofluorescent analysis of HDAC1 (green) in 3T3 cells. The cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a HDAC1 rabbit polyclonal antibody (Product # PA1-860), at a concentration of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-rabbit IgG Superclonal secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

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Experimental details: Immunofluorescent analysis of HDAC1 in C6 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a HDAC1 polyclonal antibody (Product # PA1-860) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). HDAC1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of HDAC1 in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a HDAC1 polyclonal antibody (Product # PA1-860) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). HDAC1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of HDAC1 in MCF-7 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a HDAC1 polyclonal antibody (Product # PA1-860) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552). HDAC1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

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Experimental details: Immunolocalization of HDAC1 in HeLa cells using Product # PA1-860.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human pancreas tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a Rabbit Polyclonal Antibody recognizing HDAC1 (Product # PA1-860) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a Rabbit Polyclonal Antibody recognizing HDAC1 (Product # PA1-860) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human heart tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a Rabbit Polyclonal Antibody recognizing HDAC1 (Product # PA1-860) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Figure 3. Total HDAC activity in extracts of the developing rostral corpus callosum. (A) Total HDAC activity was measured in protein extracts from anterior corpus callosum at p1, p5, p8, p11, and p24 using an acetylated fluorimetric substrate (black bars). The experiments were then repeated by incubating the same samples with TSA (white bars), or with sirtinol (hatched bars). (B) The protein levels of distinct HDAC isoforms were assessed by Western blot analysis on the same tissue extracts. Blots were probed with antibodies against class I (HDAC-1, -2, -3, and -8) and class II (HDAC-4, -5, -6, and -7), and actin as loading control.

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Experimental details: Figure 4. Cellular and temporal profile of expression of class I and II HDACs in maturing OLs. Confocal images of the medial part of the body of the corpus callosum at rostral levels, identified in coronal brain sections from p5 (A-H) and p24 (I-P) neonatal rats, after staining with antibodies for HDAC-1 to -8 (A-P, green) and for CC1 (A-P, red). Bar, 20 mum. 63x objective, LSM510 confocal microscope (Carl Zeiss MicroImaging, Inc.).

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Experimental details: Figure 5. Cell specificity of class I HDAC expression during the first postnatal week of development. Immunohistochemistry of coronal brain sections from p5 rat pups stained with antibodies against HDAC-1, -2, -3, and -8 (A-L, green), CC1 (A, D, G, and J, red), NeuN (B, E, H, and K, red), and GFAP (C, F, I, and L, red). Bar, 20 mum. 25x objective, DM RA (Leica). Each immunostaining was performed at least three times from three distinct p5 rat brains.

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Experimental details: Fig. 3 Loss of NURD subunits in HGPS cells (A) Immunofluorescence staining of control and HGPS primary dermal fibroblasts with the indicated antibodies. (B) Quantitative analysis of the fluorescence intensity signal in control and HGPS cells. N>200; Values represent averages +- S.D. from three experiments. (C) Western blot with the indicated antibodies on total cell lysates prepared from dermal fibroblast from two unaffected individuals and two HGPS patients. (D) HDAC activity measured in total lysates or in HDAC1 immunoprecipitates prepared from primary fibroblasts. Values represent averages +- S.D. from three experiments. Scale bar: 10 um.

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Experimental details: Figure 2 EGF enhances nuclear and cytoplasmic survivin protein levels . INS-1 cells were serum-deprived overnight then treated with or without EGF for 4 hours. (A) Cells were fixed and immunostained with a survivin antibody, stained with DAPI (DNA detection) then analyzed by Z-stack confocal microscopy. (B) Nuclear and cytoplasmic protein fractions were prepared, resolved by SDS-PAGE then transferred to PVDF membranes and immunoblotted with anti-survivin, anti-actin, and anti-HDAC1.

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Experimental details: Figure 4 HDAC activity directly regulates the expression of Sox11. A. Quantitative RT-PCR was performed to validate changes of transcript levels for transcriptional regulators, cell cycle regulators and myelin specific genes in OPCs either untreated or treated with TSA for one day. The data reflect the results of qPCR results performed in duplicate from 2-4 independent biological replicates (* p

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Experimental details: Figure 3 MTM affects class 1 HDAC expression levels. Real time RTPCR shows that MTM (300 nM) does not significantly affect HDAC1 ( A ) and HDAC3 ( C ) expression levels; whereas it significantly reduces HDAC2 ( B ) and HDAC8 ( D ) expression levels. These changes covary with neuroprotection by MTM as the non-protective analog PreB (300 nM) has no effect on the expression of these genes. Cells were treated for five hours and the experiments were repeated three times. ( E ): Western Blots show that MTM treatment reduces the protein levels of class 1 HDACs. ( F ): Quantification of three different western blots reveals that only HDAC2 and HDAC3 protein levels are significantly affected by MTM treatment. ( G ): Unlike sodium butyrate (5 mM) and TSA (0.66 muM), MTM does not induce acetylation of histone H3. For the western blot experiments, cells were treated with the compounds for 48 hours and experiments were repeated three times. Statistical analysis was conducted by 1way ANOVA followed by the Dunnett post test. Significant levels compared to control treatment alone. *: p < 0.05 and **: p < 0.01 and ***: p < 0.0001.

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Experimental details: Figure 4 MTM affects class 1 HDAC expression levels. ( A ) Western blot analysis reveals that Myc knockdown using ShRNA significantly affects class I HDAC protein levels. ( B ) Quantification of Western blots. Statistical analysis was conducted by 1way ANOVA followed by the Dunnett post test. Significant levels compared to control shRNA alone. * p < 0.05; and ** p < 0.01; and *** p < 0.0001.

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Experimental details: Figure 1. Conditional ablation of HDAC1 and HDAC2 in retinal ganglion cells. (a) The SLICK-H transgenic mouse line uses the Thy-1 gene regulatory elements to drive coexpression of YFP and a tamoxifen-activatable Cre fusion protein. Schematic map of Hdac1 and Hdac2 alleles depicts the location of loxP sequences. Upon Cre-mediated recombination after tamoxifen injection, the genomic region located between the loxP sites is excised, thereby inactivating the conditional Hdac1 and Hdac2 alleles. (b) Transverse section of the retina of transgenic mice without tamoxifen injections shows expression of YFP (green) in different retinal layers. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. All retrogradely Fluorogold-labeled retinal ganglion cells (yellow) colocalize with YFP (green; right panel). Scale bar: 20 um (c) Retinal immunofluororescence analysis demonstrates loss of HDAC1/2 in the YFP-expressing cells of the retinal ganglion cell layer after tamoxifen injection in conditional knockout mice. Scale bar: 20 um.

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Experimental details: Figure 3 Quiescent state of differentiated cells entails the assembly of RBL2-E2F4-HDAC1-BRM repressor complex at PARP1 promoter and the HDAC1-BRM induced chromatin compaction. The effect of RBL2 silencing on PARP1 expression was determined with western blot ( a ) and real-time PCR ( b ) in human blood-derived monocytes 72 h after cell transient transfection with siRNA. Transcription regulating epigenetic marks ( c , d , e ) as well as HDAC1 ( f ), histone H3 density ( g ) and BRM ( h ) at PARP1 promoter were analyzed with ChIP. The interaction among repressive complex components was confirmed with immunoprecypitation ( i ) by pulling down HDAC1 and detection of co-immunoprecipitated proteins with western blot. In order to verify the contribution of HDACs ( j , k ) and BRM ( l , m ) in PARP1 repression blood-derived monocytes were incubated with the corresponding inhibitors (iHDAC - sodium butyrate - 0.5 mM and iBRM/iBRG1 - PFI-3 - 1 uM) for 24 h. PARP1 expression was monitored at the protein level with western blot ( j , l )), while PARP1 mRNA was determined with real-time PCR ( k , m ). The effect of iHDAC on HDAC1, RBL2 and BRM association with chromatin ( n , o , p ) and the chromatin compaction (H3 density) in differentiated cells was assayed with ChIP ( r ).

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Experimental details: Figure 4 PRC2 facilitates PARP1 silencing by associating with RB1-E2F1-HDAC1-BRM/BRG1 repressor complex in G1 arrested cells. Epigenetic modifications ( a , b and d ), recruitment of HDAC1 ( c ), EZH2 ( e ), BRM ( g ), BRG1 ( h ) to PARP1 promoter and the density of histone H3 ( f ) were compared by ChIP between proliferating cells and cells arrested in G1 phase by their treatment with mimosine (Mim, 200 muM) for 48 h. Composition of PARP1 repressive complex in G1-arrested cells was determined by pulling down HDAC1 and followed by WB detection of RB1, E2F1, EZH2, BRM and BRG1 ( j ). The expression of PARP1 in HSPC supplemented with mimosine (50 u) and additionally with 5 mM iHDAC, 125 nM iEZH2 and the mixture of 5 mM iHDAC and 125 nM and iEZH2 for 24 h was analyzed with western blot ( k ) and real-time PCR ( l ). Expression of PARP1 in G1-arrested cells treated with BRM/BRG1 inhibitor (iBRM/iBRG1 - PFI-3 - 1 muM) was determined with real-time PCR ( n ). Changes in epigenetic marks and in protein composition in PARP1 promoter upon THP-1 cells treatment with iHDAC/iEZH2/Mim versus Mim alone were monitored with ChIP ( n - t ).

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Experimental details: Figure 3 HDAC1 dysregulation in FTLD-TDP Tg mice A Top, representative Western blot data of HDAC1 and TDP-43 in extracts obtained following RIPA fractional extraction in the frontal cortices and hippocampus from 6 months old of FTLD-TDP or WT mice. Bottom, semi-quantification of HDAC1 and TDP-43 expression levels. N = 5 mice per group, data are presented as mean +- SEM (%), **** P < 0.0001 by multiple t -tests. B Top, representative Western blot data of HDAC1 and TDP-43 in the cytosolic and nuclear extracts. Bottom, quantification of HDAC1 and TDP-43 levels. N = 5 mice per group, data are presented as mean +- SEM (%), *** P = 0.0005 (TDP-43) or 0.0004 (HDAC1); **** P < 0.0001 by multiple t -tests. C IF staining of HDAC1 and SMI-32 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Scale bar: 150 mum. Blocked area in left bottom picture is showed in a magnified view in the right bottom picture. Scale bar: 50 mum. D Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6 months old of FTLD-TDP and WT mice. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. N = 5 mice per group, data are presented as mean +- SEM (%), * P = 0.0415 by t -test. E Left, representative Western blot data of nuclear acetylated-histone H3 (Lys 9/14) and total histone H3 in 6 months old of WT and FTLD-TDP Tg mice. Right, quantification of nuclea

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Experimental details: Figure 5 TDP-43 interacts with HDAC1 and traps HDAC1 in inclusion bodies A Left panel: Flag-tagged full-length HDAC1 was overexpressed with myc-tagged TDP-43 in HEK-293T cells; the cell lysates were immunoprecipitated for flag and immunoblotted for TDP-43 and flag. Right panel: myc-tagged TDP-43 was overexpressed with flag-tagged full-length HDAC1 in HEK-293T cells; the cell lysates were immunoprecipitated for myc and immunoblotted for flag and TDP-43. B Upper left: Flag-tagged full-length HDAC1 (b.I) or various truncation mutations (b.II-IV) were overexpressed with myc-tagged TDP-43; the catalytic domain is indicated in red. Lower panel: the Western blotting of cell lysates immunoprecipitated for flag and immunoblotted for TDP-43. C Upper panel: Immunoprecipitation of cytosolic HDAC1 and immunoblotting of HDAC1 and TDP-43 in WT and FTLD-TDP Tg mice. Lower histogram: Quantification of immunoprecipitation results of HDAC1 and TDP-43 in WT and Tg mice. N = 5 mice per group, data are presented as mean +- SEM (%), * P = 0.0149, *** P = 0.0003 by t -test. D Western blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group. Source data are available online for this figure.

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Experimental details: Figure 7 Pharmaceutical gain of HDAC1 activity repairs aberrant cell cycle activity, DNA damage, and neuronal loss A Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8 months old of mice were separated from total tissue lysates, were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * P = 0.0203 (WT versus Tg-Veh) or 0.0408 (Tg-Veh versus Tg+5104434) by multiple comparison. B Representative data of Western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * P = 0.0105 or *** P = 0.0008 by multiple comparison. C Representative data of Western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** P = 0.0022 (Tg)/0.0012 (Tg+5104434) by multiple comparison. D Left panel: representative data of Western blot for cell cycle-related and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice versus Tg+Vehicle mice, # Tg+5104434 mice versus Tg+Vehicle mice. Statistical analysis by multiple t -test with FDR correction, Q = 1%. ** P

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Experimental details: Fig. 4 Interaction candidates are coexpressed with TBX2 in the pulmonary mesenchyme and interact in HEK293 cells. a Co-immunofluorescence analysis of candidate interaction partners (red) and TBX2 (green) on frontal sections of the right lung of E14.5 Tbx2 cre/ + embryos. Antigens are color-coded and nuclei were counterstained with DAPI (blue). Insets or selected regions in overview images are magnified in rows 2,4 and 6. b In situ proximity ligation assay of TBX2 and candidate interaction partners on 10 um frontal sections of E14.5 wildtype and Tbx2 cre/fl mutant lungs. Direct interaction is visualized by small red fluorescent dots. Larger more diffuse orange stains are due to auto-fluorescence of blood cells. Nuclei are counterstained with DAPI (blue). c Western blot analysis of co-immunoprecipitation experiments for verification of TBX2 interaction with candidate proteins on 10% SDS polyacrylamide gels. Detection was performed with an anti-TBX2 primary antibody and developed with chemoluminescence-IHC. Arrows indicate TBX2 bands. Lanes were loaded as follows: No antibody: IP without specific antibody resembling negative IP-control; 5% input: 5% of crude cell extract before precipitation; empty: no protein loaded; IP: co-immunoprecipitate with antibody for specific candidate. Expected molecular weight for TBX2.HA approx. 76.2 kDa

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Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 7 HDAC1 expression is reduced in adult OPCs by alcohol exposure. (A) Immunostaining of HDAC1 showing HDAC1 + GFP + OPCs (arrowheads) was remarkably reduced in the cortex of alcohol-treated mice. The dotted white box was magnified in the right panel. Scale bar 50 mum. (B) Quantification of HDAC1 + GFP + OPCs in the corpus callosum and prefrontal cortex (* p < 0.05, *** p < 0.001, n = 6).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Glutamate and TNFalpha induce cytoplasmic localization of HDAC1 in hippocampal neurons in culture. A , Primary cultures of rat hippocampal neurons (12 DIV) either untreated (control) or treated with 50 mu m glutamate and 200 ng/ml TNFalpha (Glut/TNFalpha) for 1 h and stained with DAPI and antibodies against HDAC1 (green) or NFM (red). B , Primary cultures of rat hippocampal neurons treated the same way as described in A and stained with DAPI and antibodies against HDAC1 (green) or nonphosphorylated NFH (SMI32, red), a marker of axonal damage. Scale bars, 25 mum.