LNCaP cells were plated at 2–4 × 105 cells/well in 6-well plates, allowed to adhere, and treated at ~70% confluence with increasing amounts of A-485rs with or without 20 μM I-CBP112 at a constant 0.07% DMSO for 24 h. After washing with PBS, cells were harvested by lysis in 2x SDS loading buffer [100 mM Tris-HCl (pH6.8), 20% glycerol, 4% SDS, 50 mM EDTA, 2% β-mercaptoethanol, and 0.04% bromophenol blue]. Lysate was heated to 95°C for 10 min and equal amounts separated on 15% tris-glycine gels. Equal loading was confirmed by total H3 histone blotting. Total H3 blots were probed with 1:5000 anti-total histone H3 (Abcam 1791) in 5% BSA-TBST for 1 h followed by anti-rabbit HRP at 1:5000 for 1 h at room temperature in 2.5% BSA-TBST. H3K18ac blots were probed overnight at 4°C with 1:1000 H3K18ac (Millipore 07–354) in 2.5% BSA-TBST followed by anti-rabbit HRP as above. The average relative acetylation of quadruplicate biological replicates was determined and a representative Western blot is shown.
Anti total histone h3
Manufactured by Abcam
Sourced in United States
Anti-total histone H3 is a primary antibody that recognizes the total histone H3 protein, a core component of nucleosomes. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and quantify the total histone H3 levels in different cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
H3k18ac, by Merck Group (1 mentions)
Anti h3k9me2, by Cell Signaling Technology (1 mentions)
Anti ehmt1, by Abcam (1 mentions)
Anti mouse or anti rabbit secondary antibodies, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Anti glun2b, by Abcam (1 mentions)
Anti ac h3, by Abcam (1 mentions)
3 protocols using anti total histone h3
1
Acetylation Dynamics in LNCaP Cells
2
Quantitative Analysis of EHMT1 Histone Methylation
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Normal and EHMT1+/‐ mutant human dermal fibroblasts were lysed in 4× Laemmli buffer (250 mm Tris (pH 6.8), 20% glycerol, 8% SDS, 0.0025% bromophenol blue, 1 mm β‐mercaptoethanol), collected by scraping with a rubber policeman and then sonicated for 10s. Samples were boiled at 95 ⁰C for 5 min then subjected to 14% SDS polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes and probed with the following primary antibodies: anti‐H3K9me2 (Cell Signaling Technology, Danvers, MA, USA), anti‐total Histone H3 (Abcam, Cambridge, MA, USA) in 3% bovine serum albumin in TBST, or anti‐EHMT1 (Abcam, Cambridge, MA, USA) in 5% milk. Anti‐mouse or anti‐rabbit secondary antibodies (Millipore, Danvers, MA, USA) were incubated on the membranes, after three successive washes with TBST, for 1 h at room temperature, followed by detection of bands with ECL (Pierce, Rockford, IL, USA). Quantification of bands was done using ImageJ (Rasband).
3
Hippocampal Protein Expression Analysis
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One week after the brain tissue was removed, proteins were extracted from the right hippocampus as previously described (Wang et al., 2016). An equal amount of protein (20 mg) was extracted from each hippocampus and separated by 4–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk, followed by 4°C incubation overnight with anti-total histone H3 (rabbit, polyclonal antibody, 1:1000; Abcam, Cambridge, MA, USA), anti-Ac-H3 (rabbit, polyclonal antibody, 1:1000; Abcam), anti-CBP protein (rabbit, polyclonal antibody, 1:1000; Abcam), and anti-GluN2B (rabbit, polyclonal antibody, 1:1000; Abcam). In addition, GAPDH (rabbit, polyclonal antibody, 1:1000; Abcam) was used as an internal control. After a series of processes (more detail can be found in our previously published paper (Wang et al., 2013), the optical density values of the target bands of each group were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the results were presented as the ratio between the intensity of the target proteins and GAPDH.
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