Fig. 7: Screening of key residues involved in the polyubiquitination of RACK1. | Communications Biology

Fig. 7: Screening of key residues involved in the polyubiquitination of RACK1.

From: Nematode serine protease inhibitor SPI-I8 negatively regulates host NF-κB signalling by hijacking MKRN1-mediated polyubiquitination of RACK1

Fig. 7: Screening of key residues involved in the polyubiquitination of RACK1.

a Site mutation of lysine (K183 or/and K185) with arginine (K183R, K185R, or K183R + K185R) and expression of RACK1 in HEK 293T cells. b, c The influence of site mutation of RACK1 on the relative nuclear p65 level and relative luciferase activity of NF-κB in HEK 293T cells. The gray values are analysed using an Image J software. Error bars are shown as mean ± standard error of mean (SEM). d The influence of K183R, K185R, K212R and K225R mutations of RACK1 (RACK1-AM) on the relative mRNA levels of A20, IL-8, IκBα and TNF-α in HEK 293T cells. e–h Mutations of R183K, R185K, R212K and R225K RACK1-AM resume the negative effects of RACK1 on nuclear p65 level, NF-κB, relative mRNA levels of A20, IL-8, IκBα and TNF-α in HEK 293T cells. Cells transduced with empty vector are used as negative control. Tubulin and Lamin B1 are used as internal control of cytoplasmic and nuclear proteins, respectively. Cells transduced with empty vector are used as negative control. * p < 0.05; ** p < 0.01; **** p < 0.001; **** p < 0.0001.

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