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. 2022 Mar 9;7:71. doi: 10.1038/s41392-022-00888-1

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© The Author(s) 2022, corrected publication 2023

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Fig. 4 — Effect of SHK on the NEMO/IKKβ complex. a HEK293T cells were co-transfected with eukaryotic plasmids expressing full-length Flag-NEMO and HA-IKKβ, and then treated with SHK. Whole-cell extracts were collected and subjected to a Co-IP assay and western blotting using antibodies against Flag and HA. b Prokaryotic plasmids were used to express truncated MBP-IKKβ (701–745) and GST-NEMO (1–196) in E. coli. SHK was pre-incubated with IKKβ and NEMO, respectively. The interaction of NEMO/IKKβ was analyzed by Co-IP and western blotting using antibodies against MBP and GST. c SHK was pre-incubated with 1 μM GST-NEMO for 30 min, and injected to determine the binding of GST-NEMO to MBP- IKKβ on the chip surface. d The inhibitory effects of SHK were quantified to calculate the IC₅₀. e SHK was pre-incubated with 0.5 μM MBP- IKKβ for 30 min, and injected to determine the binding of MBP- IKKβ to GST-NEMO on the chip surface. f The inhibitory effects of SHK were quantified to calculate the IC₅₀. g, h After stable transfected with IKKβ (g) or NEMO (h) shRNA or nonspecific control shRNA with lentivirus infection, LoVo cells were incubated with or without SHK (1 μM). Cell index curves were generated in real time using an xCELLigence instrument