Cells were lysed by RIPA buffer and added the bromophenol blue loading buffer, and then the samples were boiled for 10 min and centrifuged at 12,000 rpm for 5 min. The whole-cell lysate was separated into 8% SDS-acrylamide gels and transferred to PVDF membranes. After that, the membranes was blocks by 5% milk in TBST and probed with primary antibodies including mouse SETD3 (3B3, generated by Wuhan Dia-An Company), rabbit polyclonal SETD3 (Abclonal, A8071), MyoD1 (Proteintech, 18943-l-AP), HDAC1 (Abclonal, A2238), Cyclin E1 (Cell Signaling Technology, 20808 S), Myogenin (Abcam, ab124800; or Santa Cruz, D-10, sc-13137), MHC (Developmental Studies Hybridoma Bank, MF-20), β-Actin (Proteintech, 6008-I-Ig), and α-Tubulin (Sigma, T9026). For generation of mouse monoclonal SETD3 antibody, His-tagged full-length human SETD3 protein was expressed in E. Coli and purified as described previously14 (link). Purified His-SETD3 proteins were immunizated into 5-8 weeks old Balb/C mice and boosted additional 4 times. After several steps including hybridoma production, screening, cloning, and expanding the hybridomas, a subclone named 3B3 was validated and amplified followed the procedure described as before39 (link). Membranes were further probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and the protein bands were visualized using chemiluminescence detection reagents.
Hdac1
Manufactured by ABclonal
HDAC1 is a histone deacetylase enzyme that plays a role in the regulation of gene expression by removing acetyl groups from histone proteins. It is involved in the epigenetic control of chromatin structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Myogenin, by Abcam (1 mentions)
α tubulin, by Merck Group (1 mentions)
Myod1, by Proteintech (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Txnip, by Cell Signaling Technology (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Canada balsam, by Merck Group (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bx43 light microscope, by Olympus (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 680 goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 790 goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
3 protocols using hdac1
1
Western Blot Analysis of Muscle Differentiation Markers
2
Histological and Immunofluorescence Analysis
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The harvested tissues were fixed with 4 % formalin buffer (PFA) and proceeded to the paraffin-embedded block preparation. The sections (5 μm) were subject to the de-waxing in xylene and rehydration with gradient ethanol (from 100 % to 50 %). For the H&E staining, slides were incubated with haematoxylin for 5 min and subsequently 0.25 % eosin for another 5 min. The slides then were immersed in Canada balsam (Sigma-Aldrich, USA) and followed the observation with a BX43 light microscope (Olympus, Japan). For immunofluorescence, after rehydration, the slides were subject to antigen retrieval step with 10 mM citrate buffer and then followed by blocking with 10 % goat serum for 1 h at room temperature. The slides were incubated with primary antibodies Ki67 (ab15580, Abcam), TXNIP (#14715, Cell signalling), HDAC1 (A18304, Abclonal) for overnight at 4 °C and subject to the corresponding secondary antibodies with Alexa Fluor 488 (A32723, Invitrogen), Alexa Fluor 568 (A-11031, Invitrogen) respectively, and DAPI (D1306, Invitrogen) for the staining of nucleus. Finally, the slides were mounting with fluorescent mounting medium (Dako, Denmark). The fluorescent signals were captured by using the confocal microscope (Carl Zeiss LSM 780, Germany).
3
Epigenetic Regulation Assay using Antibodies
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The commercial antibodies used in this study and their sources are as follow: histone H3 (ab1791), UBC9 (ab33044), Sin3A (ab129087), histone H3 phospho S10 (ab14955), γH2AX (ab81299), lamin B1 (ab16048) and lamin A/C (ab133256) from Abcam; SUMO-1 (ET1606-53) and SUMO-2/3 (ET1701-17) from HUABIO; H3K4me3 (07-473) from Sigma-Aldrich; HDAC1 (A0238) and HDAC2 (A2084) from ABclonal; p62 (CY9081) from Abways; RARP (#9542), Caspase-3 (#9662) from CST; FLAG, HA, and GAPDH from Abmart. RbAp46 and acetylated H3 antibodies were homemade. The following secondary antibody were used: Alexa Fluor 680 goat anti-rabbit IgG (Jackson ImmunoResearch, 111-625-144); Alexa Fluor 790 goat anti-mouse IgG (Jackson ImmunoResearch, 115-655-146); Alexa Fluor 594 goat anti-rabbit IgG (Jackson ImmunoResearch, 111-585-003); Alexa Fluor 488 goat anti-mouse IgG (Jackson ImmunoResearch, 115-545-003). ML-792 (HY-108702) was purchased from MCE.
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