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H2bub

Manufactured by Merck Group
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H2Bub is a lab equipment product manufactured by Merck Group. It is designed to detect and measure the level of histone H2B monoubiquitination, a post-translational modification that plays a role in various cellular processes.

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Lab products found in correlation

Ab28629, by Abcam (2 mentions) Supersignal west pico, by Thermo Fisher Scientific (2 mentions) H2aub k119, by Cell Signaling Technology (2 mentions) Supersignal west femto, by Thermo Fisher Scientific (2 mentions) Nanog, by Santa Cruz Biotechnology (1 mentions) β catenin, by Santa Cruz Biotechnology (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Carboplatin, by Merck Group (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Topo 2, by Santa Cruz Biotechnology (1 mentions) Ub p4d1, by Santa Cruz Biotechnology (1 mentions) Mab052, by Merck Group (1 mentions) Bard1 h 300, by Santa Cruz Biotechnology (1 mentions) Ps15 p53, by Cell Signaling Technology (1 mentions) γh2ax, by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Gapdh antibody, by Merck Group (1 mentions) Myc tag antibody, by Santa Cruz Biotechnology (1 mentions) Flag tag antibody, by Merck Group (1 mentions) Phosphor gsk3β, by Cell Signaling Technology (1 mentions)

7 protocols using h2bub

1

Immunoblot Analysis of Histone Modifications

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Nuclear lysates were prepared as previously described31 (link). Nuclear extracts were separated on either 8%, 10% or 20% SDS-PAGE gels. Blots were probed with primary antibodies (H2A-Ub[K119] [Cell Signaling Technology], H2A [Cell Signaling Technology], H2B-ub [EMD Millipore], H2B [Cell Signaling Technology #12364, clone D2H6], H3 [Cell Signaling Technology], H4 [Cell Signaling Technology], HA [Cell Signaling Technology #2367, clone 6E2], TRIM37 [Abcam ab95997, or custom made by 21st Century Biochemicals against a synthetic peptide corresponding to amino acids 444–460 of the human protein followed by affinity purification] or], RNF2 [Abcam ab28629], and α-tubulin [in-house]) overnight at 4°C, washed five times in TBS plus 0.1% Tween (TBST) and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Membranes were washed five times in TBST and visualized on autoradiography film after incubating with ECL reagent (Supersignal West Pico or Supersignal West Femto; Thermo Scientific). Immunoblots were quantified using Image J software version 1.47v (NIH).
Bhatnagar S., Gazin C., Chamberlain L., Ou J., Zhu X., Tushir J.S., Virbasius C.M., Lin L., Zhu L.J., Wajapeyee N, & Green M.R. (2014). TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature, 516(7529), 116-120.
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2

Immunoblot Analysis of Histone Modifications

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Nuclear lysates were prepared as previously described31 (link). Nuclear extracts were separated on either 8%, 10% or 20% SDS-PAGE gels. Blots were probed with primary antibodies (H2A-Ub[K119] [Cell Signaling Technology], H2A [Cell Signaling Technology], H2B-ub [EMD Millipore], H2B [Cell Signaling Technology #12364, clone D2H6], H3 [Cell Signaling Technology], H4 [Cell Signaling Technology], HA [Cell Signaling Technology #2367, clone 6E2], TRIM37 [Abcam ab95997, or custom made by 21st Century Biochemicals against a synthetic peptide corresponding to amino acids 444–460 of the human protein followed by affinity purification] or], RNF2 [Abcam ab28629], and α-tubulin [in-house]) overnight at 4°C, washed five times in TBS plus 0.1% Tween (TBST) and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Membranes were washed five times in TBST and visualized on autoradiography film after incubating with ECL reagent (Supersignal West Pico or Supersignal West Femto; Thermo Scientific). Immunoblots were quantified using Image J software version 1.47v (NIH).
Bhatnagar S., Gazin C., Chamberlain L., Ou J., Zhu X., Tushir J.S., Virbasius C.M., Lin L., Zhu L.J., Wajapeyee N, & Green M.R. (2014). TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature, 516(7529), 116-120.
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3

Ovarian Cancer Cell Characterization

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Fallopian tube epithelial cells (FTSEC or FTEC) were used as normal ovarian cells (generously provided by Dr. Amir Jazaeri) [13 ]. OV90 and SKOV3 cells were purchased from ATCC, isogenic A2780 (cisplatin-sensitive) and A2780/CP70 (cisplatin-resistant) cells were previously described [14 (link)]. OV90 and SKOV3 cells were cultured in 1:1 DMEM/F12 (Mediatech). FTSEC, A2780 and A2780/CP70 cells were cultured in RPMI [14 (link)]. All media were supplemented with 10% FBS and 1x Penicillin/Streptomycin. Carboplatin was from Sigma and Rad6 expression vector was obtained from Addgene [15 (link)]. The siRNAs used in this study were purchased from Dharmacon and the transfections were done using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Antibodies specific to the following proteins were used: Gli1 (Cell Signaling Technology); GAPDH, ALDA1H1, BMI1, Nanog, OCT4, Myc, and β-Catenin (Santa Cruz Biotechnology); Rad6 (Bethyl Laboratories); H2B, H3K79me3 and SOX2 (Abcam); and Ub-H2B (Millipore).
Somasagara R.R., Tripathi K., Spencer S.M., Clark D.W., Barnett R., Bachaboina L., Scalici J., Rocconi R.P., Piazza G.A, & Palle K. (2015). Rad6 upregulation promotes stem cell-like characteristics and platinum resistance in ovarian cancer. Biochemical and biophysical research communications, 469(3), 449-455.
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4

Immunoprecipitation and Western Blotting Analysis

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Total protein (100 μg) from the indicated NEs was immunoprecipitated as described previously (7 (link), 63 (link)). Immunoprecipitations (IPs) were performed with antibodies against CstF-50 (Bethyl), Ub (P4D1; Santa Cruz Biotechnology), HA (conjugated beads from Sigma), and p97 (Bethyl). Western blot analysis was done with antibodies against histones (MAB052; Millipore), RNAP IIO (H5; Covance), BRCA1 (sc-1021; Santa Cruz Biotechnology), BARD1 (H-300; Santa Cruz Biotechnology), Ub-H2A (Millipore), Ub-H2B (Millipore), and Topo II (Santa Cruz).
Fonseca D., Baquero J., Murphy M.R., Aruggoda G., Varriano S., Sapienza C., Mashadova O., Rahman S, & Kleiman F.E. (2018). mRNA Processing Factor CstF-50 and Ubiquitin Escort Factor p97 Are BRCA1/BARD1 Cofactors Involved in Chromatin Remodeling during the DNA Damage Response. Molecular and Cellular Biology, 38(4), e00364-17.
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5

Chromatin Immunoprecipitation of H2Bub

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shGFP and shEny2 CH12 cells were stimulated for 24 hr with CIT. ChIP was performed using the Magna ChIP Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. In brief, 5 × 106 cells were fixed in the presence of 1% formaldehyde for 10 min at room temperature. The reaction was stopped by the addition of glycine to a final concentration of 0.125 M. A soluble chromatin fraction containing fragmented DNA of 200–500 bp was obtained after cell lysis and sonication. IP was performed by incubating the lysate with 5 μg of H2Bub (Millipore) antibody. The immunoprecipitated DNA was subjected to detection by qPCR normalized to the amount of input followed by the maximum value in each data set. Primers used to amplify a sequence upstream of Sμ can be found in Table S1.
Ramachandran S., Haddad D., Li C., Le M.X., Ling A.K., So C.C., Nepal R.M., Gommerman J.L., Yu K., Ketela T., Moffat J, & Martin A. (2016). The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation. Cell reports, 15(7), 1554-1565.
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6

Chromatin Immunoprecipitation Antibody Protocol

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γH2AX (Millipore), H2Bub (Millipore), H2B (Abcam), Kap1-pS824 (Cell Signaling), p53-pS15 (Cell Signaling), AID (Cell Signaling), Eny2 (Santa Cruz), and β actin (Sigma) antibodies were used as specified by the manufacturers’ protocols.
Ramachandran S., Haddad D., Li C., Le M.X., Ling A.K., So C.C., Nepal R.M., Gommerman J.L., Yu K., Ketela T., Moffat J, & Martin A. (2016). The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation. Cell reports, 15(7), 1554-1565.
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7

Western Blot Analysis of Protein Expressions

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Cells or tissue samples were lysed in RIPA buffer with protease inhibitor cocktail (Sigma) and phosphatase inhibitors PhosSTOP (Roche). The extracts were dissolved in Laemmli buffer and boiled at 95 °C for 5 min. The lysates (20–30 μg of total protein) were separated with SDS-PAGE gels, transferred to PVDF membrane (BIO-RAD) then blotted with primary and secondary antibodies. The primary antibodies used were anti-Usp22 antibody (Abcam or homemade as described previously) [15 (link)], Flag-tag antibody (Sigma or Cell Signaling Technology), Myc-tag antibody (Santa Cruz), Gapdh antibody (Millipore, Burlington, MA, USA), FSP-1 (Proteintech, Rosemont, IL, USA), H2Bub (Millipore) and cytokeratin 8 (Developmental Studies Hybridoma Bank). Total Akt antibody and phosphor-Akt(S473), total p44/42 (ERK1/2), phosphor-p44/42, GCN5, phosphor-GSK3β, GSK3β, GRB2, E-cadherin and H2B antibodies were from Cell Signaling Technology. ATXN7L3 was a gift from Dr. Tora [40 (link)].
Kuang X., McAndrew M.J., Mustachio L.M., Chen Y.J., Atanassov B.S., Lin K., Lu Y., Shen J., Salinger A., Macatee T., Dent S.Y, & Koutelou E. (2021). Usp22 Overexpression Leads to Aberrant Signal Transduction of Cancer-Related Pathways but Is Not Sufficient to Drive Tumor Formation in Mice. Cancers, 13(17), 4276.
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