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. Author manuscript; available in PMC: 2022 Oct 5.
Published in final edited form as: Sci Signal. 2022 Jul 12;15(742):eabm5995. doi: 10.1126/scisignal.abm5995

Fig. 2. USP26 colocalizes with RNF12 and interacts with the RNF12 basic region.

Fig. 2

(A) Immunofluorescence localization of FLAG-tagged human USP26 (FLAG-hUSP26) and FLAG-tagged mouse USP26 (FLAG-mUSP26) coexpressed with HA-mRNF12 in Rlim-/y mESCs. Nuclei were stained with Hoechst. Scale bar, 20 μm. Data are representative of n = 3 independent experiments. (B) Immunofluorescence localization of endogenous USP26 in Usp26+/y and Usp26Δ/y mESCs and RNF12 in Rlim+/y and Rlim-/y mESCs. Nuclei were stained with Hoechst. Scale bar, 20 μm. Data are representative of n = 2 independent experiments. (C) Immunoblotting (IB) for USP26 and RNF12 in size-exclusion chromatography fractions of Rlim+/y mESC extracts. Data are representative of n = 3 independent experiments. (D) Immunoblotting for USP26 and RNF12 in Rlim+/y mESCs fractionated into soluble cytoplasmic and nucleoplasmic (TUBB3) and chromatin-associated (phospho-Ser10 Histone H3, pH3) fractions. ERK1/2 is a loading control. Data are representative of n = 3 independent experiments. (E) Immunoblotting for FLAG and HA in HA immunoprecipitates (IP) from Rlim-/y mESCs expressing the indicated combinations of empty vector (-), FLAG-hUSP26, and HA-mRNF12. Data are representative of n = 3 independent experiments. (F) Immunoblotting for USP26, HA, and ERK1/2 in immunoprecipitates from endogenous HA-tagged RNF12 knock-in (HA-RNF12 KI) mESCs. Immunoprecipitation with IgG is a negative control. Data are representative of n = 3 independent experiments. (G) mRNF12 deletion mutants used for mapping the interaction with hUSP26. In the 4xSA mutant, Ser residues at positions 212, 214, 227, and 229 were mutated to Ala. (H) Immunoblotting for FLAG and HA in HA immunoprecipitates from Rlim-/y mESCs expressing empty vector (control) or FLAG-hUSP26 with the indicated HA-tagged mRNF12 deletion mutants. Data are representative of n = 3 independent experiments.