mouse monoclonal anti o glcnac  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Time-resolved multiomics profiling reveals chromatin O-GlcNAc modification promotes senescence-associated transcriptional program

doi: 10.1038/s41467-025-68143-z

Figure Lengend Snippet: a Representative SA-β-gal staining of LU-RAS and SK-RAS cells treated with Doxy to undergo OIS for the indicated times. Scale bars, 200 μm. b Flow cytometry was used to determine the percentages of cells in the G1, S, and G2 phases. LU-RAS and SK-RAS cells were treated with Doxy to undergo OIS for the indicated times. c Samples of LU-RAS and SK-RAS cells undergoing OIS were collected on D0, D2, D4, D6, D8, and D10 for time-course analysis. Total cell lysates and crosslinked chromatin complexes were isolated and subjected to immunoblot analysis. O-GlcNAc modification was detected using a CTD110.6 O-GlcNAc-specific antibody. d – g LU-RAS and SK-RAS cells were treated with shRNA targeting OGT, an OGT overexpression construct (pcDNA-OGT), OSMI-4, TMG, or an empty vector control and induced to undergo OIS for 4 days (D4) in the presence of Doxy. d Representative SA-β-Gal staining images are shown. Scale bars, 200 μm. e Representative fluorescence images of EdU incorporation and Hoechst staining in LU-RAS cells undergoing OIS at D4. Scale bars, 100 μm. Cells undergoing OIS and induced to progress to D4 were subsequently pulsed with EdU for 2 h, followed by Hoechst staining. f – g Total cell lysates were extracted and subjected to immunoblot analysis. O-GlcNAc modification was detected using a CTD110.6 O-GlcNAc-specific antibody. The data are presented as means ± SD. The p values were determined using a two-tailed unpaired Student’s t -test. For ( a , b ) and ( d , e ), n = 4 biologically independent experiments were performed with similar results. The immunoblots shown in ( c , f – g ) are representative of three biologically independent experiments with similar results. The source data for the graphs in ( a , b , d , e ) as well as the blots in ( c , f , g ) are provided in the File.

Article Snippet: After thorough washing to remove unbound antibody, the membrane was incubated with HRP-conjugated secondary antibodies at for 1 h. The primary anti-bodies used were anti-O-GlcNAc CTD110.6 (CST, #9875, 1:1000), Ras G12V (CST, #14412, 1:1000), anti-JUN (Abcam, #ab40766, 1:1000), anti-GATAD2A (CST, #17705, 1:1000), anti-OGT (Abcam, #ab96718, 1:1000), anti-OGA (CST, #60406, 1:1000), anti-GAPDH (CST, #5174, 1:4000), anti-Flag (CST, #14793, 1:1000), anti-HA (CST, #3724, 1:1000), anti-Histone 3 (CST, #4499, 1:4000), MBD2 (Abcam, #ab188474, 1:1000), MTA1 (Proteintech, #30545-1-AP, 1:1000), CHD4 (Proteintech, #14173-1-AP, 1:1000), SMCA4 (Proteintech, #21634-1-AP, 1:1000), SMRC1 (Proteintech, # 17722-1-AP, 1:3000), SNF5 (Abcam, #ab307985, 1:1000), p53 (Proteintech, #10442-1-AP, 1:4000), p21 WAF1/Cip1 (Proteintech, #10355-1-AP, 1:1000), p16 INK4A (Proteintech, # 10883-1-AP, 1:1000), LMNB1 (Proteintech, #12987-1-AP, 1:5000), MKI67 (Proteintech, #84192-4-RR, 1:5000), SP1 (Proteintech, #21962-1-AP, 1:5000), SRSF2 (Proteintech, #20371-1-AP, 1:5000), β-actin (Proteintech, #20536-1-AP, 1:5000), TERT (Proteintech, #27586-1-AP, 1:500), FOXK1 (Proteintech, # 29338-1-AP, 1:2000).

Techniques: Staining, Flow Cytometry, Isolation, Western Blot, Modification, shRNA, Over Expression, Construct, Plasmid Preparation, Control, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Time-resolved multiomics profiling reveals chromatin O-GlcNAc modification promotes senescence-associated transcriptional program

doi: 10.1038/s41467-025-68143-z

Figure Lengend Snippet: a STRING PPI network and functional enrichment analysis of epigenetic transcriptional OCPs in LU-RAS cells during OIS progression. The node fill colors represent the proteomic quantification of OCPs (fold change at D10 relative to D0; red: high, blue: low). b Immunoblot analysis of chromatin complexes isolated from LU-RAS cells at OIS time points D0 and D10. c Heatmap showing the chromatin binding sites and TF enrichment derived from motif analysis of upregulated COGC-seq-annotated promoter peaks in LU-RAS cells during OIS. The dot color represents the degree of TF motif enrichment, and the dot size reflects the percentage of target sequences with TF-binding sites. P values were calculated using a hypergeometric test. Multiple testing correction was performed using the Benjamini–Hochberg method. d Whole-cell lysates of LU-RAS cells at specified OIS time points were immunoprecipitated with an anti-O-GlcNAc antibody ( EPR19847 ), and the levels of NuRD and SWI/SNF components were analyzed by immunoblotting. e LU-RAS cells at OIS time points D0 and D10 were treated with OSMI-4 or vehicle. Whole-cell lysates were subjected to immunoprecipitation with an anti-GATAD2A or anti-JUN antibody. Immunoprecipitates were analyzed by immunoblotting to detect O-GlcNAc (CTD110.6) and the presence of NuRD or SWI/SNF complex components. The immunoblots are representative of three biologically independent experiments that yielded similar results ( b , d , e ). The source data for the graphs in ( c ) and the blots in ( b , d , e ) are provided in the File.

Article Snippet: After thorough washing to remove unbound antibody, the membrane was incubated with HRP-conjugated secondary antibodies at for 1 h. The primary anti-bodies used were anti-O-GlcNAc CTD110.6 (CST, #9875, 1:1000), Ras G12V (CST, #14412, 1:1000), anti-JUN (Abcam, #ab40766, 1:1000), anti-GATAD2A (CST, #17705, 1:1000), anti-OGT (Abcam, #ab96718, 1:1000), anti-OGA (CST, #60406, 1:1000), anti-GAPDH (CST, #5174, 1:4000), anti-Flag (CST, #14793, 1:1000), anti-HA (CST, #3724, 1:1000), anti-Histone 3 (CST, #4499, 1:4000), MBD2 (Abcam, #ab188474, 1:1000), MTA1 (Proteintech, #30545-1-AP, 1:1000), CHD4 (Proteintech, #14173-1-AP, 1:1000), SMCA4 (Proteintech, #21634-1-AP, 1:1000), SMRC1 (Proteintech, # 17722-1-AP, 1:3000), SNF5 (Abcam, #ab307985, 1:1000), p53 (Proteintech, #10442-1-AP, 1:4000), p21 WAF1/Cip1 (Proteintech, #10355-1-AP, 1:1000), p16 INK4A (Proteintech, # 10883-1-AP, 1:1000), LMNB1 (Proteintech, #12987-1-AP, 1:5000), MKI67 (Proteintech, #84192-4-RR, 1:5000), SP1 (Proteintech, #21962-1-AP, 1:5000), SRSF2 (Proteintech, #20371-1-AP, 1:5000), β-actin (Proteintech, #20536-1-AP, 1:5000), TERT (Proteintech, #27586-1-AP, 1:500), FOXK1 (Proteintech, # 29338-1-AP, 1:2000).

Techniques: Functional Assay, Western Blot, Isolation, Binding Assay, Derivative Assay, Immunoprecipitation