Fig. 2: Huwe1 forms complex with both Brpf3 and Myst2 and ubiquitinates Myst2. | Cell Death & Differentiation

Fig. 2: Huwe1 forms complex with both Brpf3 and Myst2 and ubiquitinates Myst2.

From: BRPF3-HUWE1-mediated regulation of MYST2 is required for differentiation and cell-cycle progression in embryonic stem cells

Fig. 2

a Coomassie blue staining of proteins associated with Brpf3. Specific Brpf3-interacting proteins were purified by immunoprecipitation using anti-FLAG affinity gel and identified by MS analysis. Identified proteins are indicated on the right. b Interaction of the Huwe1-Brpf3-Myst2 complex was analyzed by co-immunoprecipitation using Myst2 antibody. Co-immunoprecipitation was performed in MG132 treated mESCs. Immunoprecipitates were analyzed by western blot with anti-Myst2, anti-Brpf3, and Huwe1 antibodies. c Simultaneous interaction between Huwe1, Brpf3, and Myst2 was confirmed by sequential immunoprecipitation. In first immunoprecipitation, interaction was determined using anti-FLAG affinity gel, and then following second immunoprecipitation was carried out with eluted immunoprecipitates from first immunoprecipitation using Brpf3 antibody. d Protein expression of Brpf3 and Myst2 in Huwe1-overexpressing cells were investigated by western blot analysis. E14tg2a cells were transfected with plasmids expressing pCAG-HA (empty vector) and pCAG-HA-Huwe1. Alpha-tubulin was used as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. e Protein expression of Brpf3 and Myst2 in Huwe1 depleted cells were investigated by western blot analysis. Alpha-tubulin was used as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. f Protein expression of Myst2 in Huwe1 inhibited mESCs was analyzed by western blot using Huwe1 inhibitor (BI8622). Indicated amounts of BI8622 was treated for 24 h to inhibit Huwe1. Alpha-tubulin was used as the loading control. g Ubiquitination of Myst2 in Huwe1-overexpressing cells was analyzed by immunoprecipitation. pCAG-FLAG and pCAG-FLAG-Huwe1 were co-transfected with HA-tagged Ubiquitin in 293T cells and treated with 10 μM MG132 for 6 h. Protein lysates were immunoprecipitated with anti-Myst2 antibody and detected by western blot with anti-FLAG, anti-Myst2, and anti-HA antibodies.

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