ZFIN Image: Huang et al., 2022, Fig. 5

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Fig. 5

AKT signaling promotes RNF12-mediated SMAD7 ubiquitination.

A Immunoblot analysis of whole-cell lysate and immunoprecipitations derived from HA-Ub-expressing HEK293T cells transfected with Flag-RNF12 WT, RNF12 S215A, or RNF12 S347A/T349A. B AKT signaling regulates RNF12-mediated ubiquitination of SMAD7. Immunoblot analysis of whole-cell lysate and immunoprecipitations derived from HA-Ub-expressing HEK293T cells transfected with Flag-SMAD7 and Myc-RNF12, treated with IGF-1 (200 ng/mL) or MK2206 (5 μM) for 8 h. C RNF12 mutations attenuated ubiquitination of SMAD7 by RNF12. Immunoblot analysis of whole-cell lysates and immunoprecipitation derived from HA-Ub-expressing HEK293T cells transfected with Flag-SMAD7 and Myc-RNF12 mutants. D RNF12 S215A mutation attenuated interaction between RNF12 and SMAD7. HEK293T cells were co-transfected with Flag-SMAD7 and Myc-RNF12 wild-type or mutant plasmids. At 48 h post-transfection, cell lysates were harvested and subjected to anti-FLAG immunoprecipitation followed by anti-Myc western blotting. Protein expression was confirmed by western blotting using total cell lysates.

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