For western blot, whole wounds were collected from mice and ground using a tissue homogenizer to obtain a homogeneous protein suspension. Protein suspensions were then standardized for protein concentrations using a Bradford protein assay (BioRad). Equal amounts of protein were mixed with loading buffer and subjected to 4%-12% SDS gel electrophoresis under reducing conditions. Proteins were then transferred to nitrocellulose membranes and probed with primary antibodies (anti-mouse Setdb2, Xor (Abcam, Inc, RRID:AB_11154903), Ifnb (Abcam, Inc, RRID:AB_297431), ∥1 β (R&D Systems, RRID:AB_2049773)) diluted 1:500 in 5% non-fat dried milk powder in Tris-buffered saline with Tween buffer. Nitrocellulose membranes were then washed and incubated with HRP-secondary antibody (Cell Signaling, Inc) and visualized with timed chemiluminescence (Thermofisher Scientific).
Setdb2
Manufactured by Abcam
Sourced in United Kingdom
Setdb2 is a protein-coding gene that encodes a histone methyltransferase enzyme. This enzyme catalyzes the mono-, di- and trimethylation of lysine 9 on histone H3, which is involved in the regulation of gene expression and chromatin organization.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Bio-Rad (1 mentions)
Timed chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Setdb1, by Abcam (1 mentions)
Cd271, by Abcam (1 mentions)
P mtor, by Abcam (1 mentions)
H3k27me3, by Active Motif (1 mentions)
Branson sonifier 450, by Emerson (1 mentions)
Stat1, by Abcam (1 mentions)
Rabbit polyclonal igg, by Merck Group (1 mentions)
H3k9me3, by Abcam (1 mentions)
3 protocols using setdb2
1
Western Blot Analysis of Wound Healing
2
Comprehensive Protein Expression Analysis
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CD271, SETDB1, SETDB2, p-mTOR and Actin antibodies were purchased from Abcam, (CBG, UK). Phosphorylated AKT-Ser473 (pAKT), total AKT (T-AKT), phosphorylated ERK1,2-Thr202/Tyr204 (p-ERK1,2), total ERK (ERK1,2) antibodies were obtained from Cell Signalling Technology, (MA, USA). Antibodies for H3K4me3, H3K9me3 and H3K27me3 were purchased from Active Motif (CA, USA). Alexa Fluor 594 antibody and Hoechst 33342 were obtained from Thermo Fisher Scientific (MA, USA).
3
Chromatin Immunoprecipitation of Transcription Factors
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A total of 1 x 107 BM-Mϕ were treated with a vehicle control or cytokine for 24 hours and ChIP was performed as previously described [24 (link)]. DNA was fragmented by sonication using a Branson Sonifier 450 (Branson Ultrasonics). For immunoprecipitation, the following antibodies were used: IRF7 (Santa Cruz Biotechnology), STAT1 (Abcam), H3K9me3 (Abcam), Setdb2 (Dr. Yali Dou, University of Michigan), and rabbit polyclonal IgG (Millipore). DNA was assessed by RT-PCR using custom primers (S2 Table ).
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