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. 2020 Dec 7;55(5):629–647.e7. doi: 10.1016/j.devcel.2020.09.025

Figure 3.

Figure 3

SRPK Phosphorylation of RNF12 Regulates Nuclear Anchoring and E3 Ubiquitin Ligase Activity

(A) RNF12 localization in wild-type knockin (WT-KI), SR-motif phosphorylation site knockin (4xSA-KI), or SR-motif deletion (ΔSR-KI) mESCs was determined by immunofluorescence. Scalebar: 20 μm (Left). Quantification of the Nucleus/cytosol fluorescence intensity ratio (Right). Data represented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparisons test; confidence level 95%. (∗∗∗∗) p < 0.0001.

(B) RNF12 4xSA-KI mESCs were treated with 30 nM leptomycin B for 6 h and RNF12 localization analyzed by immunofluorescence. Scale bar: 20 μm (Top). Quantification of the nucleus/cytosol fluorescence intensity ratio (Bottom). Data represented as mean ± SEM Unpaired Student’s t test, two-sided, confidence level 95%. (∗∗∗∗) p < 0.0001.

(C) FLAG-tagged SRPK1 and SRPK2 were expressed in mESCs and localization of SRPKs and RNF12 analyzed by immunofluorescence. Scale bar: 20 μm (Left). Quantification of the cytosol/nucleus fluorescence intensity ratio (Right). Data represented as mean ± SEM Unpaired Student’s t test, two-sided, confidence level 95%. (∗∗∗∗) p < 0.0001.

(D) WT, Rlim−/y, RNF12 WT-KI, 4xSA-KI, ΔSR-KI, and W576Y-KI mESCs were treated with 10 μM MG132 for 6 h and RNF12-REX1 co-immunoprecipitation analyzed. RNF12, REX1 and ERK1/2 were detected by immunoblotting. (∗) indicates non-specific signal.

(E) REX1 levels were analyzed in RNF12 WT-KI, 4xSA-KI, ΔSR-KI, and W576Y-KI mESCs by immunoprecipitation followed by immunoblotting. ERK1/2 levels were detected by immunoblotting.

(F) REX1 half-life was determined in RNF12 WT-KI, 4xSA-KI, ΔSR-KI, and W576Y-KI mESCs by immunoblotting. (Top) quantification of HA-REX1 protein levels normalized to ERK1/2 and calculated protein half-life (Bottom). Data represented as mean ± SEM (n = 3). Related to Figure S3.