anti h2bub1 antibodies  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: Structural insights into the Bre1–Lge1 and RNF20/RNF40–WAC interactions critical for H2B ubiquitination

doi: 10.1093/nar/gkaf1514

Figure Lengend Snippet: The Bre1–Lge1 interface is crucial for the H2BUb1 catalysis. ( A ) Bre1 catalyzes the H2BUb1 reaction in vitro . Western blot analysis of H2B is presented. ( B ) Effects of Lge1 substitutions on the H2BUb1 catalysis in vitro . ( C ) Quantification of the reactions. The H2BUb1 amount is calculated by dividing the intensity of the H2BUb1 band with the intensity of the H2B band at the beginning of the reaction. Band intensities were read with ImageJ ( https://imagej.net ). The average and standard deviation of three independent experiments (black dots) are presented. The P -values are derived from the two-tailed Student’s t -test. ( D ) Effects of the K532E substitution in Bre1 on the H2BUb1 catalysis in vitro . ( E ) Western blot probing the effects of Bre1 and Lge1 substitutions on the levels of H2BUb1, Bre1, and Lge1 in vivo . ( F ) Western blot probing the effects of complementary charge-reversal substitutions on H2BUb1 production in vivo .

Article Snippet: Following chromatin shearing on a Diagenode Bioruptor, immunoprecipitations were carried out with anti-FLAG (14793S, Cell Signaling Technology, RRID: AB_2 572 291, 1:200 diluted) or anti-H2BUb1 antibodies (5546, Cell Signaling Technology, RRID: AB_10 693 452, 1:200 diluted).

Techniques: In Vitro, Western Blot, Standard Deviation, Derivative Assay, Two Tailed Test, In Vivo

Journal: Nucleic Acids Research

Article Title: Structural insights into the Bre1–Lge1 and RNF20/RNF40–WAC interactions critical for H2B ubiquitination

doi: 10.1093/nar/gkaf1514

Figure Lengend Snippet: The Bre1–Lge1 interface is crucial for H2BUb1-regulated DNA damage responses and repair. ( A ) Effects of Bre1 or Lge1 knockout or their substitutions on cell survival in the presence of DNA-damaging agents. ( B ) Schematic of the ectopic recombination assays. HO cs, HO cleavage site; HO cs-inc, inactive HO cleavage site. (C, D) Results of the ectopic recombination experiments carried out with tGI354 (C) or MK203 cells (D) . The average and standard deviation of three independent experiments (hollow dots) are presented. The P -values are derived from the two-tailed Student’s t -test.

Article Snippet: Following chromatin shearing on a Diagenode Bioruptor, immunoprecipitations were carried out with anti-FLAG (14793S, Cell Signaling Technology, RRID: AB_2 572 291, 1:200 diluted) or anti-H2BUb1 antibodies (5546, Cell Signaling Technology, RRID: AB_10 693 452, 1:200 diluted).

Techniques: Knock-Out, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: LDOC1 connects histone H2B monoubiquitination to tumor cell plasticity in non-small cell lung cancer

doi: 10.1186/s12964-025-02607-z

Figure Lengend Snippet: LDOC1 regulates H2Bub1 abundance and histone turnover through interactions with H2B/H2Bub1 and the proteasome. a–b Confocal immunofluorescence of A549 cells showing LDOC1 (red) colocalized with H2B (green, a) or H2Bub1 (green, b); nuclei are counterstained with DAPI (blue). Right, colocalization scatter plots with Pearson’s correlation coefficient (R). c–d PLA detecting in situ interactions between LDOC1–H2B (c, green puncta) or H2Bub1 (d, red puncta) in A549 cells; nuclei: DAPI. e–f Immunoblotting of whole-cell lysates ( e ) and nuclear histone extracts ( f ) from A549 sublines with LDOC1 knockdown (shLDOC1-1/-2) and matched controls, and from H1299 cells with ectopic LDOC1 expression. GAPDH (whole-cell lysates) and histone H3 (histone extracts) served as loading controls; for histone extracts, equal cell numbers were processed to ensure comparable loading. g Representative H&E and IHC staining for LDOC1 and H2Bub1 in two lung adenocarcinoma cases. h Co-immunoprecipitation (CoIP) followed by WB showing association between LDOC1 and PSMA1 in A549 cells; input and IgG controls are shown. i PLA signals indicating LDOC1–PSMA1 interactions in A549 cells; nuclei, DAPI. j–k IP of H2B ( j ) or H2Bub1 ( k ) from A549 cells with or without LDOC1 knockdown and with or without BTZ (20 nM, 30 min), followed by WB to detect polyubiquitinated species. Whole-cell lysates combined with histone extracts were precleared with control IgG and immunoprecipitated with the indicated antibodies; immunoprecipitates were probed with anti-ubiquitin, anti-H2B, and anti-H2Bub1. Microscopy: Panels a–d and i were acquired on a Leica Stellaris 8 confocal microscope at 400 × ; scale bars, 10 μm

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LDOC1 (custom-made); anti-H2B (Abcam, ab1790; WB, IP, IF); anti-H2Bub1 (ubiquityl-Histone H2B [Lys120], Cell Signaling Technology, #5546; WB, IF, ChIP); anti-GAPDH (GeneTex, GTX100118) and anti-THAP12 (Bethyl, A300-586 A; WB, IF, IP); anti-PSMA1 (Abnova, H00005682-M01; WB, IF, IP); FITC-conjugated anti–α-tubulin (Sigma-Aldrich, F2168; IF); anti-ubiquitin (Cell Signaling Technology, #3936; WB); and anti–E-cadherin (Cell Signaling Technology, #3195; WB, IF).

Techniques: Immunofluorescence, In Situ, Western Blot, Knockdown, Expressing, Immunohistochemistry, Immunoprecipitation, Control, Ubiquitin Proteomics, Microscopy