rabbit anti-origin recognition complex subunit 2 (orc2  (ABclonal Biotechnology)

Journal: Science Advances

Article Title: FBXO42 facilitates Notch signaling activation and global chromatin relaxation by promoting K63-linked polyubiquitination of RBPJ

doi: 10.1126/sciadv.abq4831

Figure Lengend Snippet: ( A ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding epigenetically modified proteins. Then, cell lysates were incubated with S-protein beads and blotted with antibodies against FLAG- or MYC-epitope tags. ( B ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding SWI/SNF complex proteins. Then, the cells were harvested and analyzed as described in (A). ( C ) Heatmap showing the differential interaction between chromatin factors and RBPJ in WT and FBXO42 KO cells as identified by MS. ( D ) Enrichment analysis of the differentially interacting proteins of heterochromatin components is shown on the basis of GO annotation. ( E ) Immunofluorescence detection of HP1α foci in WT and FBXO42 KO cells. Scale bars, 10 μm. ( F ) HP1α foci number and percentage of HP1α foci area were calculated using ImageJ software. ( G ) WT and FBXO42 KO cells were digested with micrococcal nuclease (MNase) for 3 min, and chromatin relaxation was monitored by the release of nucleosomes. ( H ) Chromatin association of the SWI/SNF subunits SMARCA2, SMARCA4, and SMARCC2 in WT and FBXO42 KO cells was analyzed using Western blotting after chromatin isolation. ORC2 served as the loading control. ( I ) WT and FBXO42 KO cells were digested with deoxyribonuclease (DNase) for 3 min and followed with agarose gel electrophoresis analysis. ( J ) Chromatin from WT and FBXO42 KO cells was isolated, and DNase I was digested and used as substrate for accessibility assay. ( K and L ) The heatmap view for ATAC-seq signal intensity at TSSs in WT and FBXO42 KO JURKAT cells. ( M ) ATAC-seq peaks; H3K4m1, H3K4m3, and H3K27ac ChIP-seq peaks; and DNase-seq peaks downloaded from ENCODE database at MYC locus were analyzed. (A), (B), and (E) to (J), n = 3. Quantitative data are presented as means ± SEM. P values were calculated using two-tailed Student’s t tests. * P < 0.05 and ** P < 0.01.

Article Snippet: The following primary antibodies were used: rabbit anti-RBPJ [5313S, Cell Signaling Technology (CST), RRID:AB_2665555], mouse anti-FBXO42 (TA800283, OriGene, RRID:AB_2625356), THE hemagglutinin (HA) Tag (A01244, GenScript), THE c-Myc Tag (A00704, GenScript), ANTI-FLAG M2 antibody (B3111, Sigma-Aldrich, RRID:AB_2910145), rabbit anti-ubiquitin (AF0306, Beyotime), rabbit anti–β-actin (AC026, ABclonal, RRID:AB_2768234), rabbit anti-LSD1 (YM0422, ImmunoWay), rabbit anti-SMARCA4 (ET1611-85, HUABIO), rabbit anti-SMARCA2 (ER65406, HUABIO), rabbit anti-SMARCC2 (ER62787, HUABIO), and rabbit anti-origin recognition complex subunit 2 (ORC2) (A15697, ABclonal).

Techniques: Construct, Modification, Incubation, Immunofluorescence, Software, Western Blot, Isolation, Agarose Gel Electrophoresis, ChIP-sequencing, Two Tailed Test