anti-h3t3ph antibody  (GL Biochem)

Journal: bioRxiv

Article Title: A time-resolved atlas of histone modifications during mitotic entry

doi: 10.1101/2025.09.29.679131

Figure Lengend Snippet: A – A Pearson correlation heatmap of ChIP-sequencing of H3T3ph, H3K4me1, H3K4me2 and H3K4me3 throughout mitotic entry. B – Numbers of enriched peaks at different timepoints. Bars represent mean ± standard deviation. n=2. C – A UCSC genome browser view showing the normalized (log 2 (IP/Input)) ChIP-seq read densities of H3K4me1 (light blue), H3K4me2 (dark green), H3K4me3 (violet) and H3T3ph (dark blue) on the chromosome 1. Regions shaded in pink (red *) highlight the overlap between H3K4me1 and H3T3ph. D – A magnified view of the red rectangle region from panel C, the TSS of POU2F1 gene. E – Profile plots showing the normalized ChIP-seq signal densities of H3K4me1, H3K4me2, H3K4me3, and H3T3ph around the TSS (TSS ± 20 kb) of protein-coding genes.

Article Snippet: Cells were permeabilized with 0.2% Triton-X100 for 5 min. After washing with PBS, the cells were incubated for 1 hour with 5% BSA in PBS and then with primary antibodies, H3T3ph (16B2; mouse; diluted 1:500 in blocking buffer) and ACA (human; Antibodies Incorporated; 15-234; diluted 1:500 in blocking buffer), in 1% BSA and 0.1% Tween-20 in PBS overnight at 4°C.

Techniques: ChIP-sequencing, Standard Deviation

Journal: bioRxiv

Article Title: A time-resolved atlas of histone modifications during mitotic entry

doi: 10.1101/2025.09.29.679131

Figure Lengend Snippet: A – A Pearson correlation heatmap of ChIP-seq of H3T3ph throughout mitotic entry with H3K9me3 and H3K27me3. B – A violin plot of spike-in calibrated H3T3ph levels at the repetitive centromere of Chr8, as well as its p- and q-arms. C – The same but for Chr5 with the non-repetitive centromere. Statistics for B and C was computed by fitting a linear mixed effect model of log(enrichment). “Timepoint” and “position along the chromosome“ were fit as fixed effects, and “sample” as a random effect. Gaussian distribution of models’ residuals was confirmed. Since T-distribution is symmetric, one-sided p value was calculated for the null hypothesis of no centromeric enrichment of H3T3ph. The hypothesis was rejected in the case of a repetitive centromere on Chr8 (p < 0.0001, ****). The p-values were adjusted with Benjamini-Hochberg procedure. D – A UCSC genome browser view of the magnified centromeric region of Chr8 and Chr5. CENP-H ChIP-seq shows CENP-H (red)-binding peaks (spike-in calibrated) at the centromere of Chr8 (Left) and Chr5 (Right). Other tracks show landscape profile of H3T3ph (dark blue), H3K9me3 (dark pink) and H3K27me3 (dark orange) signal (spike-in calibrated or log 2 (IP/Input)) around the centromeric region.

Article Snippet: Cells were permeabilized with 0.2% Triton-X100 for 5 min. After washing with PBS, the cells were incubated for 1 hour with 5% BSA in PBS and then with primary antibodies, H3T3ph (16B2; mouse; diluted 1:500 in blocking buffer) and ACA (human; Antibodies Incorporated; 15-234; diluted 1:500 in blocking buffer), in 1% BSA and 0.1% Tween-20 in PBS overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay

Journal: Oncogene

Article Title: Genome-wide CRISPR screen uncovers a synergistic effect of combining Haspin and Aurora kinase B inhibition

doi: 10.1038/s41388-020-1296-2

Figure Lengend Snippet: Depletion or inhibition of Haspin sensitizes cells to VX-680 treatment. ( A ) Confirmation of Haspin depletion in Haspin KO cell lines. HCT116 wild-type and Haspin KO cells were collected and directly lysed by SDS loading buffer. The expression of Haspin, H3T3ph, and H3S10ph were detected with indicated antibodies. Anti-Tubulin and anti-H3 blots, respectively, were included as loading controls for Haspin, H3T3ph and H3S10ph blots. The arrow indicates nonspecific bands. ( B ) Cells treated with Haspin depletion or inhibition exhibit hypersensitivity to VX-680 treatment. HCT116 wild-type and Haspin KO cells were treated with CHR-6494 and VX-680 for consecutive 9 days, either in combination (VX combined with 0, 40, 80, and 120 nM CHR) or single-agent treatment (CHR; VX). Colonies were fixed, stained, and further analyzed by Image J. Experiments were performed in triplicate with duplicate biological replicates. Representative images and results are shown. Student t tests were performed to estimate differences between 2 groups. Error bar represents SE (n = 3); *** P < 0.001; **** P < 0.0001. ( C ) HCT116 cells were treated with indicated concentrations of VX-680 and CHR-6494 and further incubated for 24 h. Then cells were lysed by SDS loading buffer and examined by immunoblotting with indicated antibodies. The H3T3ph and H3S10ph levels were determined with indicated antibodies. H3 served as loading control. ( D ) HCT116 cells were incubated with VX-680 (0.03, 0.05, and 0.1 μM) and CHR-6494 (0.05, 0.1, 0.15, and 0.2 μM) in combination or single agent treatment. After 24 h, cells were lysed and examined by Western blotting with indicated antibodies. ( E ) Western blots show the effect of single-agent treatment (0.03, 0.05, and 0.1 μM VX; 0.2 μM CHR). Samples were collected and blotted as described in ( D ).

Article Snippet: Antibodies against H3T3ph (Cat. No. 13576S) and H3S10ph (Cat. No. 9701S) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing, Staining, Incubation, Western Blot, Control