Figure 2. RING1B and DDB2 cooperate in H2A ubiquitylation. (A) RING1B interacts with DDB2. Control cells and cells expressing FLAGRING1B were irradiated with UV light. After immunoprecipitation with FLAG-M2-Agarose the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (B) Endogenous immunoprecipitations with RING1B antibodies after UV irradiation. Western blots of the precipitated material were incubated with the indicated antibodies. IgG lanes show unspecific staining of the IgG heavy chains. (C) DDB2 associates with RING1B. Control cells and cells expressing FLAGDDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Epistatic relationship of RING1B and DDB2 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV dosages. Control cells were transfected with either control siRNA (control) or DDB2 siRNA (DDB2). RING1B knockdown cell lines were transfected with either control siRNA (RING1B) or DDB2 siRNA (RING1B + DDB2). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 6). (E) Knockdown of RING1B does not impair DDB2 recruitment. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative DDB2 and BMI-1 abundance was calculated. Values are given as mean ± SEM (n = 3). (F) Knockdown of DDB2 shows reduced H2A-ubiquitylation but unaltered BMI-1 recruitment. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, DDB2). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitylation and RING1B abundance was calculated. Values are given as mean ± SEM (n = 4).
Figure 2.

RING1B and DDB2 cooperate in H2A ubiquitylation. (A) RING1B interacts with DDB2. Control cells and cells expressing FLAGRING1B were irradiated with UV light. After immunoprecipitation with FLAG-M2-Agarose the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (B) Endogenous immunoprecipitations with RING1B antibodies after UV irradiation. Western blots of the precipitated material were incubated with the indicated antibodies. IgG lanes show unspecific staining of the IgG heavy chains. (C) DDB2 associates with RING1B. Control cells and cells expressing FLAGDDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Epistatic relationship of RING1B and DDB2 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV dosages. Control cells were transfected with either control siRNA (control) or DDB2 siRNA (DDB2). RING1B knockdown cell lines were transfected with either control siRNA (RING1B) or DDB2 siRNA (RING1B + DDB2). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM (n = 6). (E) Knockdown of RING1B does not impair DDB2 recruitment. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative DDB2 and BMI-1 abundance was calculated. Values are given as mean ± SEM (n = 3). (F) Knockdown of DDB2 shows reduced H2A-ubiquitylation but unaltered BMI-1 recruitment. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, DDB2). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitylation and RING1B abundance was calculated. Values are given as mean ± SEM (n = 4).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal