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. 2023 Jan 20;80(2):47. doi: 10.1007/s00018-022-04659-7

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© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

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Fig. 6 — A CHAMP1-POGZ interacting region and intact chromodomain are required for CDYL2 regulation of mitosis and genome stability. a Graphic representation of Gal4 DBD (G4)-tagged CDYL2 full-length (FL) and deletion or triple point mutants. b CHAMP1, POGZ and Gal4 immunoblot of anti-Gal4 or control IgG IP performed on nuclear extracts from Hela cells transfected with the indicated plasmids. An excerpt of the Gal4-CDYL2 blot is shown at lower exposure in the image below that of the full blot, to facilitate visualization of the Gal4-CD construct which is obscured by the antibody light chain signal in the higher exposure blot. Reactive bands of the expected molecular weight are indicated by single asterisks (*). Cross-reactive bands from the light chain of the IP antibodies in the lower panel are indicated (**). The experiment was repeated three times with similar results. c Western blot validation of CDYL2 RNAi knockdown and expression of the Gal4-CDYL2 constructs indicated above each lane. Shown are CDYL2, Gal4, and actin blots on the same membrane. Reactive bands of the expected molecular weight are indicated by single asterisks (*). d The indicated phenotypes were counted in Hela cells treated with control (siCtrl) or siCDYL2 along with an empty vector (EV) or one of the CDYL2 cDNAs indicated in (a). Cells were incubated with MG-132 for 2 h before analysis to enrich in metaphase cells. Shown is the mean of three independent assays and S.D. T test of differences between siCDYL2/EV and siCtrl/EV, or siCDYL2/EV co-transfected with one of the five rescue plasmids indicated in (a) (*p < 0.05; **p < 0.01). e PLA using CHAMP1 and CENP-B antibodies was performed on Hela cells treated with Control or CDYL2 siRNA, with co-transfection of either pcDNA3-Gal4 or Gal4-CDYL2-FL expression plasmid, as indicated. Representative images are shown. The experiment was repeated three times with similar results. f Hela cells were treated as in panels (c) and (d), fixed, and PLA assay for CHAMP1 proximity to CENP-B was performed, as in (e). Shown is the mean number of PLA dots per nucleus of three independent assays and S.D. T test compared each condition to the siCDYL2/G4 sample (***p < 0.001)