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Fig. 2 | BMC Biology

Fig. 2

From: Smad4 SUMOylation is essential for memory formation through upregulation of the skeletal myopathy gene TPM2

Fig. 2

Smad4 is SUMO-modified by PIAS1 in the hippocampus. a Co-IP experiments showing that Smad4 is associated with PIAS1 (left panel) and SUMO1 (right panel) in the rat hippocampus. b Co-IP experiment showing that PIAS1 is associated with Smad4 in the rat hippocampus. c Flag-vector, Flag-Smad4WT (with or without the addition of SUMO1 mutant protein), and different Flag-Smad4 SUMO-mutant plasmids were transfected to the rat CA1 area and SUMOylation assay was carried out 48 h later to determine Smad4 SUMOylation at Lys-113 and Lys-159 in the hippocampus. Left panel: Immunoblotted with anti-Smad4 antibody. Right panel: The membrane was stripped and further immunoblotted with anti-SUMO1 antibody. Plasmid transfection and expression were confirmed by western blotting using anti-Flag antibody. d The quantified results. n = 5 for each group, F(5,24) = 65.09, # P < 0.001; q = 19.61, # P < 0.001 comparing the Flag-Smad4WT and Flag-vector groups; q = 10.1, # P < 0.001 comparing the Flag-Smad4WT and Flag-Smad4K113R groups; q = 15.27, # P < 0.001 comparing the Flag-Smad4WT and Flag-Smad4K159R groups; q = 19.92, # P < 0.001 comparing the Flag-Smad4WT and Flag-Smad4K113RK159R groups; and q = 20.82, # P < 0.001 comparing the Flag-Smad4WT and Flag-Smad4WT + SUMO1 mutant groups, using one-way ANOVA followed by post hoc Newman–Keuls multiple comparisons. Raw data and statistics are provided as Additional file 9. e Flag-vector, Flag-Smad4WT, or Flag-Smad4K113RK159R plasmid was co-transfected with His-ubiquitin plasmid to HEK293T cells. The anti-Flag antibody was used for immunoprecipitation and the anti-His antibody was used for immunoblotting. IB immunoblotting, IP immunoprecipitation, Ub-Smad4 ubiquitinated Smad4

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