Fig. 4

FAM167A regulates NIK binding and ubiquitination through DSG1. A NF-κB luciferase reporter activity in K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs. B Immunoblot analysis of p100/p52 in K562R cells after DSG1 knockdown. C NF-κB luciferase reporter activity in K562R cells 24 h after transfection of a plasmid encoding DSG1, or both DSG1 and Erbin, with or without treatment with 2 μg/ml anti-FAM167A neutralizing antibody. D Co-immunoprecipitation analysis of DSG1, Erbin, and NIK in K562S and K562R cells. E Co-immunoprecipitation analysis of DSG1 and NIK in K562R cells after treatment with 2 μg/ml of an anti-FAM167A neutralizing antibody or an isotype control for 3 h. F Immunoblot analysis of NIK ubiquitination with an anti-ubiquitin antibody after immunoprecipitation of NIK from K562R cells treated with 2 μg/ml of an anti-FAM167A neutralizing antibody or an isotype control plus 10 μM MG132 for 3 h. G Schematic model of the FAM167A-induced noncanonical NF-κB pathway. GAPDH was used as an internal standard (B, D-F). Ub, ubiquitination. Data are representative of two (B) or three (A, C-F) independent experiments (error bars, s.d. of duplicate (B) or triplicate (A, C-E) samples). Unpaired two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001