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. 2021 Aug 31;29(2):381–392. doi: 10.1038/s41418-021-00862-4

Fig. 3. FBXW2 ubiquitination p65 at K122 site depends on p300 induced acetylation.

Fig. 3

Immunoblotting analyses were performed with the indicated antibodies. A MCF-7 cells were stably expressed shRNA-FBXW2. Cells were treated with MG132 for 8 h followed by IP and western blot. B MCF-7 cells were transfected with Flag-tagged FBXW2. After 48 h transfection, cells were treated with MG132 for 8 h followed by IP and western blot. C The indicated MEFs cells were treated with MG132 for 8 h followed by IP and western blot. D FBXW2 proteins were purified from 293T cells transfected with HA-tagged FBXW2 by IP with HA beads and elution with 3× HA peptide. p65 proteins were purified from 293T cells with Flag-tagged p65 transfected by IP with Flag beads. FBXW2 and p65 on Flag beads were added into a reaction mixture containing ATP, His-ubiquitin, E1 and E2, followed by constant mixing at 37 °C for 60 min. Immunoblotting analyse was performed. E, F The 293T cells were co-overexpressed both Flag-tagged FBXW2 and different mutants of HA-tagged p65. Immunoblotting analyse was performed. G The 293T cells were co-overexpressed both HA-tagged FBXW2 and Flag-tagged p65 (WT or K122R). After 48 h transfection, cells were treated with MG132 for 8 h followed by IP and western blot. H 293T cells transfected with different ubiquitin mutants were co-overexpressed both HA-tagged FBXW2 and Flag-tagged p65. After 48 h transfection, cells were treated with MG132 for 8 h followed by IP and western blot.